The authors thank previous and current members of the laboratory for development of the protocols. The work was supported by funding from the Canadian Institutes of Health Research (CIHR) [grant # MOP-130527] and from the Natural Sciences and Engineering Research Council of Canada [NSERC-355306-2012]. NG is recipient of a Tier II Canada Research Chair. AR holds a studentship from the training program of the Respiratory Health Research Network from the Fonds de la recherche du Qu&#233;bec-Sant&#233; (FRQS).

注:该协议是这里描述使用感染了仙台病毒（SeV载体）A549细胞。然而，该协议用 ​​于SDS-PAGE和变性PAGE也与迄今测试的所有人类和小鼠细胞类型，与各种IRF3活化刺激9,15,19,24,25刺激特别骨髓细胞。 1.感染A549细胞<ol><li>维持A549细胞在培养中有15厘米板在37℃/ 5％CO 2在20毫升含10％热灭活的胎牛血清（HI-FBS）和1％L-谷氨酰胺的F12K /火腿培养基（完全F12K /火腿中）。 注:所有用于细胞培养和处理的溶液必须是无菌的。 </li><li>在感染前24小时，吸出培养基，并用10毫升蒸馏水磷酸盐缓冲盐水（D-PBS）在室温洗细胞。 </li><li>加入1毫升0.25％的胰蛋白酶-EDTA溶液，以每块板以覆盖细胞和在37℃下3分钟。 </li><li>只要第停止孵育E细胞开始从平板上通过轻轻敲击板用一只手分离。通过加入10ml每板预热完成的F12K /火腿介质灭活胰蛋白酶，将细胞转移到15ml锥形管中。 </li><li>离心在350×g离心3分钟，在RT。除去上清液和重悬细胞沉淀于8ml完全F12K /火腿介质以得到一种均一的单细胞悬浮液。 </li><li>计数使用血球细胞。种子细胞以每60毫米平板1×10 6个细胞在4毫升预热的完整的F12K /火腿介质的密度。孵育20 - 24小时，在37℃/ 5％CO 2，在20 - 24小时，将细胞形成90％汇合单层（这对应于1.5×10 6个细胞）。 </li><li>除去培养基并用2ml无血清的F12K /火腿培养基（SFM）洗细胞。加入2毫升按每60kg毫米的钢板新鲜SFM。 </li><li>在冰上和旋涡解冻仙台病毒（SeV载体）（存储等分在-80℃）的等分试样简要。 </li><li>稀日病毒在预热SFM获得60 HAU / 100微升。通过上下吹打轻轻混合，并添加100微升每平板稀释病毒在40 HAU / 10 6个细胞进行感染。不要在非感染的对照板添加病毒。 </li><li>孵育细胞在培养箱中在37℃/ 5％CO 2。用手搅动板3或4次，或使用自动轨道或摇床上直接在培养箱放置，感染的第一个小时期间。 </li><li>在2小时后感染，加入2毫升含有20％的HI-FBS，以获得10％的HI-FBS的最终浓度的F12K /火腿培养基。 </li><li>孵育细胞在培养箱中在37℃/ 5％CO 2的额外的1个，4个和7小时达到3,6，和9小时的总感染倍。在所有这些时间点，继续执行步骤2.1。 </li></ol> 2.制备的全细胞提取物（WCE） <ol><li>取出感染的媒介。通过刮除在1ml收获细胞冰冷的D-PBS和的细胞悬浮液转移到预冷的1.5毫升离心管中。 </li><li>沉淀细胞离心16000 xg离心在4℃下20秒，小心地倒出的D-PBS的所有痕迹。 注:在此步骤中，将细胞沉淀，可直接进行蛋白质提取或快速冷冻在液氮或干冰/乙醇浴中并储存在-80℃直至溶解。 </li><li>制备含有50mM HEPES pH 7.4的，150 mM氯化钠，5mM的EDTA，10％甘油和1％的Nonidet P-40的去离子水中的溶解缓冲液（DDH 2 O的）。即席添加蛋白酶（1μg/ ml的亮抑酶肽和2微克/ ml抑肽酶）和磷酸酶抑制剂（5mM的氟化钠，1mM的活化的原钒酸钠，2mM的磷酸对硝基苯酯和10mMβ甘油pH7.5）中。 注:无抑制剂的裂解缓冲液可以储存于4℃。对于原钒酸钠激活特定协议的具体试剂/ Equipme的表中描述NT。 </li><li>重悬在70微升裂解缓冲液的细胞沉淀。通常，裂解物浓度将约为2微克/微升。 </li><li>冰上孵育20分钟。闪冻裂解物温育在液氮浴中15秒。解冻溶胞产物在室温下，直到它完全熔化并涡旋10秒。重复冷冻/融化/涡流循环3次。 <ol><li>可替代地，1分钟进行在乙醇/干冰浴冷冻步骤。 </li></ol></li><li>离心机在16000 xg离心在4℃下20分钟。将上清液转移（对应于WCE）到一个新的预冷的1.5毫升离心管中。置于冰上的WCE在任何时候。 </li><li>量化使用与所述裂解缓冲液如布拉德福德基于蛋白测定26兼容的任何蛋白质定量过程的蛋白质。 </li></ol>通过SDS-PAGE高分辨率3.分辨率WCE的<ol><li>准备3变性电泳凝胶。 <ol><li>为检测IRF3形式，倒入至少16厘米长度的两个凝胶为7.5％的丙烯酰胺/双丙烯酰胺组成的分离凝胶（37.5:1），375毫摩尔Tris-盐酸pH值8.8（RT），0.1％十二烷基硫酸钠（SDS ），1％过硫酸铵和0.1％TEMED在DDH 2 O的和4％丙烯酰胺构成的积层凝胶，62.5毫的Tris-HCl pH 6.8的（RT），0.05％SDS，1％过硫酸铵和0.1％TEMED在双蒸2 O. 警告！丙烯酰胺，TEMED和SDS是有毒和/或有刺激性。戴防护手套，并在通风橱操作。 </li><li>为SeV载体蛋白的检测，倒最少8.5厘米长，类似于在3.1.1中描述，不同之处在于在分离凝胶含有12％丙烯酰胺凝胶的特性之一的凝胶。 </li></ol></li><li>通过加入1变性在步骤2.6中得到的WCE:4（V / V）5×上样缓冲液（125毫摩尔Tris-盐酸pH为6.8（RT），10％的SDS，20％（P / V）甘油，0.0005％溴酚蓝和接着在100℃加热25％β巯基乙醇在DDH 2 O的）下进行10分钟。快速离心管，以降低形成在盖的缩合。 警告！ β巯基乙醇是由吸入有毒。戴防护手套，并在通风橱操作。 </li><li>安装在迁移装置中的凝胶。填充运行含有25mM Tris碱，0.1％SDS和192毫甘氨酸在蒸馏水（DH 2 O）的缓冲器中的上和下腔室。 </li><li>负载14微升分子量标准在每16个厘米凝胶的一个孔和7微升分子量标准中的8.5厘米凝胶的一个孔中。变性WCE的负载30微克（其制备如步骤3.2中所述）中为IRF3形式检测步骤3.1.1制备的两种凝胶的每孔。负载8 - 变性WCE的10微克（其制备如步骤3.2中所述），每孔在为SeV的分析步骤3.1.2制备的凝胶。 </li><li>运行的凝胶在30 mA的恒定电流，直到迁移前沿到达凝胶的底部。 注:迁移一般持续approximately 3小时为16厘米凝胶和45分钟为一个8.5厘米的凝胶。 </li><li>继续进行到转印到硝酸纤维素膜上（步骤5）。 </li></ol> IRF3二聚本地人-PAGE 4.分析注:此方法最初是由一群T.藤田博士27的描述。 <ol><li>制备的上（ - ）和下（+），腔室电泳缓冲液。上部腔室缓冲液由25mM的Tris-盐酸pH值8.4（RT），192毫甘氨酸和1％脱氧胆酸钠（DOC）中的双蒸2 O.下部腔室缓冲液含有25mM的Tris-盐酸pH值8.4（RT）和192毫甘氨酸在双蒸2 O. 注:上，下腔室电泳缓冲液可以储存在4℃直至使用。但是，请确保它们预温至室温进行电泳之前。 </li><li>倾至少含有7.5％的丙烯酰胺/双丙烯酰胺（37.5:1）8.5厘米长的非变性分离胶，375毫的Tris-HCl pH值8.8（RT）下，1％的弹药鎓过硫酸盐和0.1％TEMED在双蒸2 O. </li><li>运行前在40mA的恒定电流将凝胶在冰上30分钟。按运行装置成冰大约到下室电泳缓冲液的水平。重要的是，该凝胶是不是在冰上。 </li><li>在预运行，混合WCE保持在冰上用2×天然-PAGE上样缓冲液1:1（体积/体积）含125毫摩尔Tris-盐酸pH为6.8（RT），30％甘油和0.1％溴酚蓝在双蒸2 O. </li><li>负载8 - 10微克WCE（如在步骤4.4中所述制备）在紧接在预运行结束。 </li><li>运行凝胶在冰上25毫安恒定电流，如上述的预运行，直到迁移前沿到达凝胶（约40分钟）的底部。 </li></ol> IRF3种5.免疫印迹分析<ol><li>制备含25毫米的Tris-Base和192毫米甘氨酸双蒸2 O传输缓冲区在冷藏，使用前4℃下传输缓冲区。 </li><li>湿三块硝酸纤维素膜切成大小小于在包含转移缓冲液的塑料/玻璃盒凝胶稍大。表明该膜的通过切割一个角的取向。传送缓冲器可重复使用3次。存储缓冲区，在4℃使用C之间。 </li><li> Uncast凝胶（SDS-PAGE和天然-PAGE）和切断每个凝胶正确方向的一个角。 <ol><li>对于本机-PAGE在室温下孵育凝胶温和搅拌，在SDS-PAGE电泳缓冲液的至少30分钟传送到传送缓冲器之前移除的DOC。 </li></ol></li><li>孵育在转移缓冲液的凝胶（SDS-PAGE和天然-PAGE）中5 - 10分钟。 </li><li>装入每凝胶转印三明治与朝向正极（泡沫垫/滤纸/膜/凝胶/滤纸/泡沫垫）的膜的转印带盒。小心删除所有气泡夹心层之间。 </li><li>执行转移的建议by中的制造商所用的传送装置。 注:执行摇动或轨道振荡平台在接下来的步骤中所有的孵育和洗涤。 </li><li>在转移时间结束时，孵育所述膜为含有双蒸2 O 7％乙酸，40％乙醇和3％甘油中固定溶液15分钟洗涤该膜在PBS 3×5分钟（137毫摩尔NaCl，2.7毫米氯化钾，10.2毫的 Na 2 HPO 4和1.8毫KH 2 PO 4在卫生署2 O）。 警告！醋酸是有毒，有刺激性，易燃。戴防护手套和通风橱下操作。 注:固定溶液可以重复使用多次。 </li><li>对于本地-PAGE膜直接进行步骤5.11。 </li><li>在含在双蒸2 O 6.57毫红色丽春红和1％乙酸红色丽春红溶液孵育他们1分钟前在的dh 2 O快速冲洗三SDS-PAGE上的膜 注:红色的丽春解决方案可为R<html> eused数次。 </li><li>在冲洗卫生署2 O膜，直至背景是白色的，足以看出染红蛋白带。注意用铅笔标记和切口周边蛋白质的过量膜。脱色膜温育5分钟的PBS下搅拌。 </li><li>在室温下孵育或O / N的膜1小时，在4℃下在封闭溶液（含0.05％吐温20和5％脱脂干牛奶的PBS（PBS-T-牛奶）洗涤膜3×5分钟在PBS-T。 注:PBS-T中洗涤是可选的，并且仅施加稀释在含有5％牛血清白蛋白（PBS-T-BSA）（ 图1和表1）在下一步骤PBS-T抗体时严格要求。 </li><li>根据顺序在图1和表1详细描述与第一抗体孵育的四个膜（由SDS-PAGE和天然-PAGE）。在PBS-T进行5×5分钟洗涤。 </li></ol>"&gt; <img alt="图1" src="/files/ftp_upload/53723/53723fig1.jpg" /> 图1的序列的免疫印迹分析的的概略说明，以检测各种IRF3磷酸和单体/二聚体形式中需要的各SDS-PAGE和天然-PAGE凝胶。应用于从免疫印迹程序中的每个凝胶所得的膜的抗体的具体顺序进行说明。注意，抗肌动蛋白抗体用于第一至确保在应用任何其他特异性抗体前，样品的相等的加载。交替序列，用抗肌动蛋白抗体的抗 - 磷酸抗体IRF3之后被应用，也适用。剥离用于抗肌动蛋白和抗西弗抗体，或抗磷酸化IRF3因为重叠的信号大小的抗IRF3抗体之间。 <a href="https://www.jove.com/files/ftp_upload/53723/53723fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within页="1"&gt; <tbody><tr><td> 一抗 </td><td> 稀释 </td><td> 稀释缓冲液 </td><td> 孵化 </td><td> 二抗 </td><td> 评论 </td></tr><tr><td> 抗肌动蛋白 </td><td> 1 / 10,000 </td><td> PBS-T-BSA </td><td>在室温15分钟</td><td>抗小鼠</td><td>使用SDS-PAGE后， 稀释antibodyies可重复使用好几次，如果保存在4℃下在0.02％的叠氮化钠的存在。 </td></tr><tr><td> 抗IRF3-P-Ser386 </td><td> 1/200 </td><td> PBS-T-BSA </td><td> O / N在4℃ </td><td>抗兔</td><td>本地-PAGE后使用。 这是不建议重用稀释的抗体</td></tr><tr><td> 抗IRF3-P-Ser396 </td><td> 1 / 10,000 </td><td> PBS-T-BSA </td><td> O / N在4℃ </td><tD&gt;抗兔</td><td> SDS-PAGE后使用。 这是不建议重用稀释的抗体反IRF3-P-396也可自Cell Signaling。最佳稀释被定义为1 / 1,000此抗体，但也可以大量之间变化。 </td></tr><tr><td> 抗IRF3-P-Ser398 </td><td> 1 / 10,000 </td><td> PBS-T-BSA </td><td> O / N在4℃ </td><td>抗兔</td><td> SDS-PAGE后使用。这是不建议重用稀释的抗体</td></tr><tr><td> 反IRF3全长 </td><td> 1/7500 </td><td> PBS-T-乳</td><td> 3小时在室温</td><td>抗兔</td><td> SDS-PAGE后使用。抗IRF3全长抗体可用于后天然-PAGE上，但它是较不敏感的检测单体。稀释抗体可以重复使用多次，如果储存在4℃下在0.02％的叠氮化钠的存在。 </td></tr><tr><td> 抗IRF3-NE小号</td><td> 0.5微克/毫升</td><td> PBS-T-乳</td><td> 3小时在室温</td><td>抗兔</td><td>本地-PAGE后使用。稀释antibodyies可重复使用好几次，如果保存在4℃下在0.02％的叠氮化钠的存在。 </td></tr><tr><td> 反西弗 </td><td> 1/14000 </td><td> PBS-T-BSA </td><td> 3小时在室温</td><td>抗兔</td><td> SDS-PAGE后使用。稀释antibodyies可重复使用好几次，如果保存在4℃下在0.02％的叠氮化钠的存在。 </td></tr><tr><td colspan="6"> 注 :稀释和缓冲器用于HRP偶联的第二抗体需要，因为它从一个不同的公司到另一个进行优化。 </td></tr></tbody></table> 用在免疫印迹过程表1.抗体的规格。 <ol start="13"><li>孵育与辣根过氧化物酶膜（HRP）的缀合，如图1和表1详细描述的二次抗体。洗涤膜5×5分钟的PBS-T，然后在PBS中2倍5分钟以充分除去吐温的痕迹。 </li><li>孵育膜1分钟在增强的化学发光试剂足以完全覆盖所述膜的一个体积。干燥使用滤纸膜。 </li><li>将膜在一个发光图像分析仪以可视化的免疫反应条带。 <ol><li>另外，执行使用敏感的X光片的免疫反应带的检测。 </li></ol></li><li>洗3×5分钟的PBS膜。 注:在此步骤中的膜可以保持干燥。但是，如果需要进一步剥离，最好是干燥该膜之前执行剥离。膜也可以被存储为短期于PBS中。 </li><li>对于不要求与抗体孵育之间剥离膜（ 见图 1）直接转到步骤5.20。 </li><li>当需要剥离抗体（ 见图 1），孵育该膜在含有2％的SDS，62.5毫的Tris-HCl pH 6.8的（RT）和0.7％β巯基乙醇在DDH 2 O的预热的剥离溶液，在50℃之间根据常规搅拌20分钟。洗3×5分钟的PBS膜。 注:在剥离过程中搅拌是关键。汽提可以在杂交炉中进行。可替代地，汽提可以使用膜在塑料袋密封并浸渍在水浴中进行。在孵育期间的5倍 - 在这种情况下，以搅动膜4是重要的。 </li><li>孵育膜1小时，在室温或O / N在4℃下在PBS-T-牛奶。 </li><li> 根据图1重复步骤5.12到5.16。 </li></ol>

<ol>
	<li>Juang, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+activation+of+interferon+A+and+interferon+B+gene+transcription+by+interferon+regulatory+factory-3.">Primary activation of interferon A and interferon B gene transcription by interferon regulatory factory-3.</a> Proc Natl Acad Sci U S A. 95 (17), 9837-9842 (1998).</li><li>Lin, R., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+casein+kinase+II+phosphorylation+in+the+regulation+of+IRF-1+transcriptional+activity.">A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity.</a> Mol Cell Biochem. 191 (1-2), 169-180 (1999).</li><li>Grandvaux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+interferon+regulatory+factor+3+target+genes:+direct+involvement+in+the+regulation+of+interferon-stimulated+genes.">Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes.</a> J Virol. 76 (11), 5532-5539 (2002).</li><li>Thompson, M. R., Kaminski, J. J., Kurt-Jones, E. A., Fitzgerald, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+receptors+and+the+innate+immune+response+to+viral+infection.">Pattern recognition receptors and the innate immune response to viral infection.</a> Viruses. 3 (6), 920-940 (2011).</li><li>Grandvaux, N., tenOever, B. R., Servant, M. J., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interferon+antiviral+response:+from+viral+invasion+to+evasion.">The interferon antiviral response: from viral invasion to evasion.</a> Curr Opin Infect Dis. 15 (3), 259-267 (2002).</li><li>Servant, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Signaling+Pathways+Leading+to+the+Phosphorylation+of+Interferon+Regulatory+Factor+3.">Identification of Distinct Signaling Pathways Leading to the Phosphorylation of Interferon Regulatory Factor 3.</a> J Biol Chem. 276 (1), 355-363 (2001).</li><li>Qin, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+IRF-3+reveals+mechanism+of+autoinhibition+and+virus-induced+phosphoactivation.">Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.</a> Nat Struct Biol. 10 (11), 913-921 (2003).</li><li>Takahasi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+crystal+structure+of+IRF-3+and+its+functional+implications.">X-ray crystal structure of IRF-3 and its functional implications.</a> Nat Struct Biol. 10 (11), 922-927 (2003).</li><li>Mori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Ser-386+of+interferon+regulatory+factor+3+as+critical+target+for+inducible+phosphorylation+that+determines+activation.">Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.</a> J Biol Chem. 279 (11), 9698-9702 (2004).</li><li>Clement, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+IRF-3+on+Ser+339+generates+a+hyperactive+form+of+IRF-3+through+regulation+of+dimerization+and+CBP+association.">Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association.</a> J Virol. 82 (8), 3984-3996 (2008).</li><li>Saitoh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+regulation+of+interferon-regulatory+factor+3-dependent+innate+antiviral+response+by+the+prolyl+isomerase+Pin1.">Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1.</a> Nat Immunol. 7 (6), 598-605 (2006).</li><li>Wathelet, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+infection+induces+the+assembly+of+coordinately+activated+transcription+factors+on+the+IFN-beta+enhancer+in+vivo.">Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo.</a> Mol Cell. 1 (4), 507-518 (1998).</li><li>Karpova, A. Y., Trost, M., Murray, J. M., Cantley, L. C., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+regulatory+factor-3+is+an+in+vivo+target+of+DNA-PK.">Interferon regulatory factor-3 is an in vivo target of DNA-PK.</a> Proc Natl Acad Sci U S A. 99 (5), 2818-2823 (2002).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TAK1-JNK+cascade+is+required+for+IRF3+function+in+the+innate+immune+response.">The TAK1-JNK cascade is required for IRF3 function in the innate immune response.</a> Cell Res. 19 (4), 412-428 (2009).</li><li>Solis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+TBK1+and+IKKepsilon+in+lipopolysaccharide-induced+activation+of+the+interferon+response+in+primary+human+macrophages.">Involvement of TBK1 and IKKepsilon in lipopolysaccharide-induced activation of the interferon response in primary human macrophages.</a> Eur J Immunol. 37 (2), 528-539 (2007).</li><li>Soucy-Faulkner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+NOX2+and+reactive+oxygen+species+for+efficient+RIG-I-mediated+antiviral+response+through+regulation+of+MAVS+expression.">Requirement of NOX2 and reactive oxygen species for efficient RIG-I-mediated antiviral response through regulation of MAVS expression.</a> PLoS Pathog. 6 (6), e1000930 (2010).</li><li>Lin, R., Heylbroeck, C., Pitha, P. M., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-dependent+phosphorylation+of+the+IRF-3+transcription+factor+regulates+nuclear+translocation,+transactivation+potential,+and+proteasome-mediated+degradation.">Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation.</a> Mol Cell Biol. 18 (5), 2986-2996 (1998).</li><li>Yoneyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+triggering+of+the+type+I+interferon+system+by+virus+infection:+activation+of+a+transcription+factor+complex+containing+IRF-3+and+CBP/p300.">Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300.</a> EMBO J. 17 (4), 1087-1095 (1998).</li><li>Servant, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+minimal+phosphoacceptor+site+required+for+in+vivo+activation+of+interferon+regulatory+factor+3+in+response+to+virus+and+double-stranded+RNA.">Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.</a> J Biol Chem. 278 (11), 9441-9447 (2003).</li><li>Bergstroem, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+in+vivo+virus-targeted+phosphorylation+site+in+interferon+regulatory+factor-3+(IRF3).">Identification of a novel in vivo virus-targeted phosphorylation site in interferon regulatory factor-3 (IRF3).</a> J Biol Chem. 285 (32), 24904-24914 (2010).</li><li>Takahasi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ser386+phosphorylation+of+transcription+factor+IRF-3+induces+dimerization+and+association+with+CBP/p300+without+overall+conformational+change.">Ser386 phosphorylation of transcription factor IRF-3 induces dimerization and association with CBP/p300 without overall conformational change.</a> Genes Cells. 15 (8), 901-910 (2010).</li><li>Noyce, R. S., Collins, S. E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modification+of+interferon+regulatory+factor+3+following+virus+particle+entry.">Differential modification of interferon regulatory factor 3 following virus particle entry.</a> J Virol. 83 (9), 4013-4022 (2009).</li><li>McCoy, C. E., Carpenter, S., Palsson-McDermott, E. M., Gearing, L. J., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+inhibit+IRF3+phosphorylation+in+response+to+Toll-like+receptor-3+and+-4+by+targeting+TBK1+activation.">Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation.</a> J Biol Chem. 283 (21), 14277-14285 (2008).</li><li>Oliere, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTLV-1+evades+type+I+interferon+antiviral+signaling+by+inducing+the+suppressor+of+cytokine+signaling+1+(SOCS1).">HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1).</a> PLoS Pathog. 6 (11), e1001177 (2010).</li><li>Kato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+involvement+of+RIG-I+in+antiviral+response.">Cell type-specific involvement of RIG-I in antiviral response.</a> Immunity. 23 (1), 19-28 (2005).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Iwamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IRF-3/-7+kinase+and+NF-kappaB+in+response+to+double-stranded+RNA+and+virus+infection:+common+and+unique+pathways.">Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.</a> Genes Cells. 6 (4), 375-388 (2001).</li><li>tenOever, B. R., Servant, M. J., Grandvaux, N., Lin, R., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+measles+virus+nucleocapsid+as+a+mechanism+of+IRF-3+activation.">Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation.</a> J Virol. 76 (8), 3659-3669 (2002).</li><li>Bibeau-Poirier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+I{kappa}B+Kinase+(IKK)-Related+Kinases+Tank-Binding+Kinase+1/IKKi+and+Cullin-Based+Ubiquitin+Ligases+in+IFN+Regulatory+Factor-3+Degradation.">Involvement of the I{kappa}B Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation.</a> J Immunol. 177 (8), 5059-5067 (2006).</li><li>Grandvaux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Activation+of+Interferon+Regulatory+Factor+3+during+Infection+by+Paramyxoviruses+Requires+MDA5.">Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5.</a> J Innate Immun. 6 (5), 650-662 (2014).</li></ol>

The authors declare that they have no competing financial interests.

我们在这里描述该协议包括耦合到几个使用磷酸化特异性抗体来区分IRF3的单体/二聚体和phosphoformsⅠ-Ⅳ高分辨率SDS-PAGE和天然-PAGE的组合。适当的检测这些IRF3的物种是必不可少的充分体现IRF3激活特定设置。例如，活化的巨噬细胞的LPS刺激导致形成二聚体，Ser396 / 398磷酸化IRF3的呈现一个低磷酸（II型），但并不过度磷酸（形式III和IV）中，图案在SDS-PAGE 15。使用所描述的协议，该信息可以从?...

<html>在无处不在和组成性表达的转录因子，干扰素（IFN）调节因子3（IRF3）是防御病原体的第一道防线主要是通过IFNβ的诱导是关键，也可通过诱导趋化因子（CC基序）配体5（CCL5 ）和几个抗病毒蛋白，包括干扰素诱导蛋白与tetratricopeptide重复IFIT1 / 2/3 1-3。 IRF3活化已经报道以下感染与众多的病毒，或暴露于聚肌胞苷酸（聚I:C）或脂多糖（LPS）4。重要的是，研究得最多的病毒已经进化机制以逃避IRF3介导的反应，从而逃避宿主先天免疫防御5。因此，监控IRF3的激活是非常重要的，了解先天抗病毒宿主防御的分子机制，而且要确定使用病毒来抵消这种应对策略。 ENT"&gt;许多公开的报道然而提供通过IRF3靶基因诱导的监视（IFNB1和IFIT1）进行IRF3活化和/或萤光素酶报告基因检测耦合到低分辨率十二烷基硫酸钠聚丙烯酰胺凝胶电泳的仅有限分析（SDS- PAGE）IRF3的分析，然而，大量的生化研究，各种IRF3突变体和IRF3晶体结构6-11的阐明的行为的分析有助于确定该IRF3是在经受了一组连续的翻译后修饰复合物磷酸化多个站点，设置的磷酸化参与IRF3活化似乎依赖于刺激，最有可能在细胞类型，在未感染的细胞，IRF3共存含有phosphoresidues，包括Thr135和Ser173非磷酸化和低磷酸物种，在1 -198氨基酸N-末端区域6,12-14。这种低磷酸的F积累IRF3的ORM由应力诱导剂，生长因子和DNA损伤剂6诱导。丝氨酸/苏氨酸残基在含有激活域IRF3的C末端区域的磷酸化触发由病毒以下激活，聚I:C或脂多糖以细胞类型依赖性15-17。 IRF3的C末端的磷酸化涉及组织在两个主要簇，Ser385 / Ser386和Ser396 / Ser398 / Ser402 / Thr404 / Ser405不低于7不同磷酸受体位点，每一个有助于IRF3活化通过二聚化，核积累，与CREB关联结合蛋白（CBP）/ P300共激活因子，DNA结合干扰素敏感反应元件（ISRE）共有序列和靶基因9,10,17-19的转录。 Thr390的磷酸化也被认为有助于病毒诱发IRF3激活20。 IRF3的质谱分析表明，Ser386，Thr390，Ser396和Ser402残基直接phosphorylat由κB激酶ε抑制剂（IKKε）ED / TANK结合激酶1（TBK1）激酶9,10。 也需要通过泛素化和蛋白酶体介导的降解10终止IRF3活化的磷酸化的C末端残基。这个过程也依赖于磷酸化的Ser339，这是必要的丙基异构酶的Pin1 10,11的募集。至少含有磷酸化Ser339 /三百九十六分之三百八十六残留IRF3的物种被认为是过度磷酸化形式。每个站点的确切序列和功能保持的讨论10,21的问题。这是现在很清楚，激活IRF3并不代表均匀状态，但不同的活性粒子表现出明显的磷酸化或二聚化特征存在10,22。 提供IRF3活化的完整理解响应于特定的病原体，因此，有必要为characterize该活性物质种类的被诱导。诱导IRF3的靶基因，IFNB1和IFIT1，已被证明是提供了可靠的读出了IRF3激活。然而，监测这些基因的表达不IRF3的不同的激活状态之间进行区分。对IRF3的激活状态在一个特定的环境进行综合分析依赖于它的磷酸化和二聚化状态10的详细特征。磷酸化（I型），低磷酸（II型）和过度磷酸化（形式III和IV）IRF3形式6,18,23可以成功地通过减少流动性高的分辨率SDS-PAGE分析解决。单体和二聚体IRF3种可以有效地确定本地-PAGE分析。组合使用用针对不同IRF3磷酸受体位点磷酸化特异性抗体时，这些方法都大大提高。 标准协议允许的分辨率差蛋白质不允许不同IRF3磷酸化形式有​​效分离。在这里，我们详细描述了一种方法来实现的最高分辨率监视使用SDS-PAGE连接到本地-PAGE结合免疫使用完全和磷酸化抗体不同的病毒激活IRF3品种的诱导，在体内不同的激活之间的歧视IRF3的形式是基于它们在SDS-PAGE观察到的迁移率的变化进行的。此外，IRF3单体和二聚物可以通过非变性电泳加以区别。与免疫印迹这两个互补技术的结合被证明是一个负担得起的和敏感的方式来获取所有必要的信息IRF3的磷酸化介导的激活的全面分析。

<table><tbody><tr><td>F12/Ham</td><td>Life Technologies</td><td>11765-054</td><td>Warm in a 37 &amp;deg;C bath before use.</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>12483-020</td><td></td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>D-PBS</td><td>Life Technologies</td><td>14190-144</td><td>For cell culture.</td></tr><tr><td>Trypsin/EDTA 0.25%</td><td>Life Technologies</td><td>25200-072</td><td></td></tr><tr><td>Sendai virus Cantell Strain</td><td>Charles River Laboratories</td><td>600503</td><td></td></tr><tr><td>Hepes</td><td>Bioshop</td><td>HEP001</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Bioshop</td><td>SOD001.5</td><td></td></tr><tr><td>EDTA</td><td>Bioshop</td><td>EDT001</td><td></td></tr><tr><td>Glycerol</td><td>Bioshop</td><td>GLY001.1</td><td>Cut the extreminity of the tip and pipet slowly as it is very thick.</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma-Aldrich</td><td>I7771</td><td>Registred trademark corresponding to Octylphenoxy poly(ethyleneoxy)ethanol (Nonidet P-40) detergent</td></tr><tr><td>Leupeptin</td><td>Bioshop</td><td>LEU001</td><td></td></tr><tr><td>Aprotinin</td><td>Bioshop</td><td>APR600.25&amp;nbsp;</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma-Aldrich</td><td>201154</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>MP Biomedicals</td><td>159664</td><td>Activation of sodium orthovanadate 0.2 M : 1) Ajust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthotovanadate solution may vary with lots of chemical. 2) The solution is yellow at pH 10.0. 3) Boil until colorless. 4) Cool to RT. 5) Reajust the pH to 10.0 and repat steps 3-4 until the solution remains colorless and stabilizes at 10.0. Store the activated sodium orthovanadate aliquots at -20 &amp;deg;C.</td></tr><tr><td>p-nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Beta-Glycerophosphate</td><td>Sigma-Aldrich</td><td>G6376&amp;nbsp;</td><td></td></tr><tr><td>Bio-Rad Protein Assay Reagent&amp;nbsp;</td><td>Bio-Rad</td><td>500-0006&amp;nbsp;</td><td>Cytotoxic</td></tr><tr><td>Acrylamide/Bis-Acrylamide (37.5 : 1) 40%</td><td>Bioshop</td><td>ACR005&amp;nbsp;</td><td>Cytotoxic</td></tr><tr><td>Tris-Base</td><td>Bioshop</td><td>DEO701</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>LabChem</td><td>LC15320-4&amp;nbsp;</td><td>Work under fume hood. Toxic and irritant.</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Bioshop</td><td>SDS001.1&amp;nbsp;</td><td>Irritant.</td></tr><tr><td>Amonium persulfate</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>TEMED</td><td>Invitrogen</td><td>15524-010</td><td>Toxic and irritant.</td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientific</td><td>B392-5</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td>Work under fume hood. Toxic to the nervous system, mucous membranes. May be toxic to upper respiratory tract, eyes, central nervous system.</td></tr><tr><td>Glycine</td><td>Bioshop</td><td>GLN001.5</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Bioshop</td><td>SHY700&amp;nbsp;</td><td>Irritant.</td></tr><tr><td>Nitrocellulose membrane (0.45 mm)</td><td>Bio-Rad</td><td>162-0115</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Bioshop</td><td>ACE222.4</td><td>Work under fume hood. Toxic, irritant and flammable.</td></tr><tr><td>Red ponceau</td><td>Sigma-Aldrich</td><td>P3504&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911&amp;nbsp;</td><td>For PBS composition for immunoblot.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Bioshop</td><td>SPD307.5</td><td>For PBS composition for immunoblot.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P0662&amp;nbsp;</td><td>For PBS composition for immunoblot.</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td>For PBS-T-BSA composition for immunoblot.</td></tr><tr><td>Non-fat dry milk</td><td>Carnation</td><td></td><td></td></tr><tr><td>Poly sorbate 20 (Tween)</td><td>MP Biomedicals</td><td>103168</td><td>Cut the extreminity of the tip and pipet slowly as it is very thick.</td></tr><tr><td>Anti-IRF-3-P-Ser386</td><td>IBL-America</td><td>18783</td><td>Store aliquoted at -20 &lt;sup&gt;o&lt;/sup&gt;C. Avoid freeze/thaw.</td></tr><tr><td>Anti-IRF-3-P-Ser396</td><td>Home made&lt;sup&gt;19&lt;/sup&gt;</td><td></td><td>Store aliquoted at -80 &lt;sup&gt;o&lt;/sup&gt;C. Avoid freeze/thaw.</td></tr><tr><td>Phospho-IRF-3 (Ser396) (4D4G)</td><td>Cell Signaling Technology</td><td>4947s</td><td>Store at -20 &lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>Anti-IRF-3-P-Ser398</td><td>Home made&lt;sup&gt;15&lt;/sup&gt;</td><td></td><td>Store aliquoted at -80 &lt;sup&gt;o&lt;/sup&gt;C. Avoid freeze/thaw.</td></tr><tr><td>Anti-IRF-3-full length</td><td>Actif motif</td><td>39033</td><td>Store aliquoted at -80 &lt;sup&gt;o&lt;/sup&gt;C. Avoid freeze/thaw.</td></tr><tr><td>Anti-IRF3-NES</td><td>IBL-America</td><td>18781</td><td>Store aliquoted at -20 &lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>Western Lightning Chemiluminescence Reagent Plus</td><td>Perkin-Elmer Life Sciences</td><td>NEL104001EA</td><td></td></tr><tr><td>LAS4000mini CCD camera apparatus</td><td>GE healthcare</td><td></td><td></td></tr><tr><td>SDS-PAGE Molecular Weight Standards, Broad Range</td><td>Bio-Rad</td><td>161-0317</td><td>Store aliquoted at -20 &lt;sup&gt;o&lt;/sup&gt;C.</td></tr></tbody></table>

a high resolution method to monitor phosphorylation-dependent activation of irf3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

图2示出 IRF3与IRF3总抗体和IRF3-磷酸化特异性抗体对Ser396和Ser398 WCE的由高分辨率的SDS-PAGE解析之后检测出的典型的免疫印迹图像。在未刺激的A549细胞，IRF3被检测为两个频带，在50和53 kDa的在对应于所述非磷酸化（形式I）的SDS-PAGE和所述低磷酸（形式Ⅱ）IRF3的物种。曝光A549细胞以SEV为3 - 9小时导致时间依赖性移缓慢迁移过度磷酸形式III和IV，它们是从窗体井分?...

Watch this Scientific Journal Video about A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3 at JoVE.com

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

a-high-resolution-method-to-monitor-phosphorylation-dependent

Research

JoVE Journal

Biology

11.8K 次观看.  Université de Montréal. Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

视频: A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

时间分辨Förster共振能量转移测定法，用于测量人细胞中的内源性磷酸化STAT蛋白

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

免疫与感染

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

原生聚丙烯酰胺凝胶电泳免疫溴分析内源性IRF5二聚化

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Source: Bailey, E. M. et al., <a href="https://www.jove.com/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> J. Vis. Exp. (2022)
This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

用于蛋白质磷酸化分析的单分子沉降测定:一种定量细胞裂解物中蛋白质磷酸化的高通量技术

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)
This video ...