この研究はによって資金を供給された：オーストリア科学財団（FWF）によるエージング（NFN S93）、欧州委員会の統合プロジェクトMiMAGEとPROTEOMAGE、科学研究費オランダゲノミクスイニシアティブ/オランダ機構（NGI / NWOの国立研究ネットワーク、05040202と050 - 060から810 NCHA）、優秀寿命（FP6 036894）、およびゲノミクスのイノベーション指向研究プログラム（SenterNovemのEUの資金ネットワーク; IGE01014とIGE5007）。

1。プラスミド構築<ol><li>オーダーカスタムオリゴヌクレオチドDGN（ACKNWFSSLSHFVIHL 11）とdgnFS（HARTGSLACPTSSSICE）のエンコードおよびDGN / dgnFS（ 図1）とGFPの融合を得るためのpEGFP-C1ベクターにそれを連結。 </li><li> pENTR方向性TOPOクローニングキットのプロトコールに従ってPCRにより、GFP-DGNおよびGFP-dgnFSのコード配列を増幅し、pLenti6/V5方向性TOPOクローニングキット（ 図6）に進みます。 </li></ol> 2。ウイルス産生<ol><li>彼らはトランスフェクションの日に90〜95％コンフルエントになるようにT75フラスコのHEK 293FT細胞外にトランスフェクションの前日（1日目）はシード。 </li><li>トランスフェクション当日に血清および15mlファルコンで抗生物質を含まないDMEM 1.5mlにリポフェクトアミン2000試薬36μlを希釈します。別の15mlファルコンを使用し、いずれかのGFPを相補的なDNA構築物を運ぶ3μgのpLenti6/V5を薄める<html> -DGNまたはGFP-dgnFS、血清および抗生物質を含まないDMEM 1.5mlに2.5μgのエンベロープのエンコードプラスミドpMD2.Gと7.5μgのベクターバックボーンpsPAX2。 5分後、希釈したDNAは、希釈されたリポフェクトアミン2000試薬と結合されます。 </li><li>形成するために、DNA-リポフェクタミン複合体を許可するように室温で20分間混合物をインキュベートする。 </li><li> HEK 293FT細胞の培養液を除去し、培地の7ミリリットル（抗生物質なしで）慎重にそれを交換して、ミキシング用フラスコ、前後にロックして（2日目）にDNA-リポフェクタミン混合物を追加します。加湿した5％CO 2インキュベーター内で37℃で一晩フラスコをインキュベートする。 </li><li>翌日、培地を変更します（抗生物質なしの10 mlの培地、3日目）。 </li><li> 48時間後（5日目）収穫上澄み、室温で5分間、300×gで遠心分離し、0.45μmPVDFフィルターを通って上清をフィルターします。 </li></ol> 培地- DMEM（HEK 293FT） テント &quot;&gt; DMEM 10％のFBS 0.1mMのMEM NEAA 6 mM L-グルタミン 1mMのMEMピルビン酸ナトリウム 1パーセントペンストレプトマイシン（オプション） 500μg/ mlのジェネティシン（オプション） 3。ポリエチレングリコール（PEG）沈殿によるウイルス濃度<ol><li> 4℃で2時間インキュベートした上清の4つのボリュームに℃、1ボリュームのポリエチレングリコール溶液（PEG 50mMのポリエチレングリコール、41 mMのNaCl、オートクレーブは、pH = 7.2）を追加慎重にそれを反転させて20〜30分毎に混ぜる。 </li><li> 4℃で30分間1500 xgでスピンダウンホワイトペレットが見えるはずです。 </li><li>上清を吸引除去し、4℃で5分間1500 xgで再度遠心操作残りのPEG溶液を吸引します。 </li><li>媒体またはでペレットを再懸濁し、リン酸緩衝生理食塩水（PBS）でピペッティングし、ダウンしてから、20〜30秒間激しくボルテックスすることによって。ガイドラインとして1 T75フラスコとアリコートそれ100μlに、500μlを使用しています。 -80℃でウイルス℃で保存</li></ol><ol><li>うち5種×6ウェルプレートの各ウェルに10 4 U2-OS細胞トランスフェクションの前日。 </li><li>トランスフェクションの日に培地を除去し、8μg/ mlのポリブレンを（ヘキサジメトリンブロマイド）を含むDMEMを1ミリリットルでそれを交換してください。濃縮ウイルス上清1:10〜1:100に希釈し、よく一つに1μlのそれぞれに追加します。高いウイルス濃度の場合は1μL、5μLと残りのウェルのために濃縮したウイルス上清10μlを使用しています。一つは、よくポジティブコントロールとして残されます。 </li><li>次の日は、DMEM 2 mlで培地を交換してください。 </li><li>選択翌日から始まります。各ウェルに10μg/ mlのブラスチシジンを適用します。一日おきに抗生物質を含む媒体を交換してください。 </li><li>非形質導入細胞の選択を開始した後、約6-7日で死んでいる。染色のために、PBSで細胞を3回洗浄し、クリスタルバイオレット（10 mg / lのクリスタルバイオレットで細胞をカバー20％エタノール）を加え、室温で5〜10分間インキュベートする。吸引クリスタルバイオレットを（数回再利用できます）と蒸留H 2 Oで2回ウエルを洗浄し、プレートを乾燥させます。 </li><li>コロニーをカウントし、千と対応する希釈（ 図7）で乗算します。この手順では、ウイルス/ mlのトランスフェクションユニット（TU）を計算することができます。 </li></ol> 培地- DMEM（HFF-2/U2-OS） DMEM 10％のFBS 6mMのL-グルタミン 1パーセントペンのStrep 5。ヒト二倍体線維芽細胞の形質導入（この手順は、任意の細胞型にも使用できます。） <ol><li>うち5種×10 4個のヒト二倍体線維芽細胞（HDFS）6ウェルプレートに伝達前日。 </li><li> 1ミリリットルの合計で伝達エンハンサーとして8μ​​g/ mlのポリブレンとともに2つの感染多重度を使用しています。 </li><li>翌日、培地を変更します。 </li><li>時細胞がコンフルエントの70〜80％にある10μg/ mlのブラストサイジンを使用して、選択を開始します。選択が完了したら（これ以上トランスフェクトしていない細胞が死なない）細胞の増殖を開始することができ、細胞は実験のための準備が整いました。ブラストによる慢性選択がプロテアソーム活性を測定する準備ができ、安定した細胞株を過剰発現するGFP-DGNまたはGFP-dgnFS融合タンパク質を生成することができます。この手順では、細胞型の独立した非常に高いトランスフェクション効率を保証します。 </li></ol> 6。フローサイトメトリーによる測定<ol><li>うち1種×10 5個の細胞（GFP-DGNまたはGFP-dgnFSどちらを運ぶ）実験の前日。 </li><li>コントロール細胞の前で3時間プロテアソーム阻害剤を用いた測定、 例えば、100μg/ mlのLLNLを扱う。 </li><li> PBSで2回洗浄し、トリプシンの0.5mlを用いて細胞を収穫します。トリプシン処理を停止するには培地の4.5mlを使用し、15mlファルコンにセルを転送します。 </li><li>スピントン室温で5分間、300×gで彼は細胞。 </li><li> 、培地を吸引除去し、PBSに細胞を再懸濁し、37℃で5分間300 xgで再びスピンダウン℃に</li><li> FACS緩衝液（10mMのHepes、140mMのNaCl、2.5 mMのCaCl 2、pH = 7.4）中の400μlに再懸濁し、PBSを吸引除去し、FACSチューブに細胞を移す。 </li><li>フローサイトメトリーによって氷と測定試料に対して細胞を維持。細胞は4℃でより長い30分を保存するべきではありません</li></ol> 7。代表的な結果 GFP-DGN融合タンパク質は、タンパク質従ってプロテアソームを標的とする配列を担持し、すぐに分解され、それはGFP蛍光シグナルの減少に対応しています。フレームシフト突然変異体（GFP-dgnFS）このシーケンスの変異バージョンを運び、プロテアソームによって分解されない、それは、より高い緑色蛍光につながる。これらの理由から、期待の高いプロテアソーム活性を持つ若いHDFSとGFP-DGで形質導入n個のフローサイトメトリー測定の両方でと落射蛍光（ 図2AおよびB、図3）の低（6％陽性細胞）の蛍光シグナルを示す。同じHDFSは陽性細胞の39.7パーセントを表示し、GFP-dgnFSで形質導入した。プロテアソーム阻害剤LLNL（N-アセチル-L-ロイシル-L-ロイシル-L-norleucinal）で細胞を処理するには、GFP-DGNおよびGFP-dgnFS細胞（ 図2AおよびBの両方で62.9％の最高に信号を発生させた）。 老化ヒト皮膚試料におけるプロテアソーム活性の可能性低下を判定するには、若い中年と古いドナーから単離し真皮線維芽細胞は、GFP-DGNおよびGFP-dgnFSに感染していた、上記のようにし、フローによる分析前に、同じ継代数に培っメトリ。これらの実験では、若い人（11.2±0.88パーセントGFP陽性細胞）と中年のドナー（20.4±2.27パーセントGFP陽性細胞は、P = 0.003）の間にGFPシグナルで明確な増加が示す、観察されているプロテアの減少高齢ドナー7（ 図4）から得られた試料でOME活動。プロテアソーム活性のさらなる減少は最古の個体（ 図4）から単離された線維芽細胞では観察されなかった。 GFP-dgnFSの蛍光強度は、すべてのケースであった〜3年齢別グループのために高いトランスフェクション効率を示している90％（データは示さず）。 <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="図1" /> 図1。 GFP-DGNおよびGFP-dgnFSシーケンス。表示は、GFP（緑色）のpEGFP-CL1はベクトル（灰色）とDGN / dgnFSシーケンス（赤）のマルチクローニング部位の3 &#39;末端である。 <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="図2" /> 図2。 GFP-DGNおよびGFP-dgnFSヒト二倍体線維芽細胞のフローサイトメトリー分析。 A. Youngにヒト包皮線維芽細胞（HFF-2）は、緑色蛍光タンパク質を運ぶレンチウイルスベクターを感染させた（GFP）-DGN遺伝子またはGFP-dgnFS（フレームシフト）は、フローサイトメトリー（FACS CantoのⅡ、Becton Dickinson）を用いてGFP蛍光のために示され、分析、構築します。示される場合、細胞はまた、プロテアソーム阻害剤N-アセチル-L-ロイシル-L-ロイシル-L-norleucinal（LLNL）で3時間処理した。非感染細胞を対照として用いた。実験は3連で行った。Bのデータは、3つの独立した実験の代表である。数字はGFP陽性細胞の量を反映しています。 <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">拡大図を表示するにはここをクリック</a> 。 <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="図3" /> 図3。 GFP-DGNおよびGFP-dgnFS HFF-2細胞の蛍光顕微鏡分析。HFF-2ヤングは、 図2と同様に処理した。感染後9日目に、細胞はvであった蛍光および位相差顕微鏡によりisualized。 <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="図4" /> 図4。人間の皮膚の老化におけるプロテアソーム活性の変化。指示の年齢層で9つの異なるドナーからのヒト包皮線維芽細胞が最小限拡大およびGFP-DGNをコードするレンチウイルスコンストラクトで感染させた。感染後9日目に、細胞をフローサイトメトリーによって分析した。データが重複での年齢グループごとに3つのサンプル（±SE）で得た。 <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="図5" /> 図5。 DGNとdgnFSのための実験的なアプローチ。カスタムオリゴヌクレオチドはのpEGFP-C1ベクターにクローン化され、ウイルスをHEK 293FT細胞を用いて、各構築物のために生産されています。ウイルスの力価が決定される。細胞がウイルスで形質導入と展開されます。有利な治療の後、細胞を分析したフローサイトメトリーによる蛍光シグナルのために。 <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="図6" /> 図6。 pLenti GFP-DGNの地図。GFP-DGN配列を含むpLenti6/V5-DESTベクターのマップが表示されます。略語：PCMV（CMVプロモーター）、GFP-DGN（GFP-DGNのシーケンス）、P SV40（SV40初期プロモーター）、EM7（EM7プロモーター）、ブラスチシジン（ブラストサイジン耐性遺伝子）とΔU3/ 3&#39;LTR（3&#39;LTR削除されたU3領域）、SV40 PA（SV40ポリアデニル化シグナル）、アンピシリン（アンピシリン耐性遺伝子）、pUC系ORI（PUC由来）、PRSV / 5&#39;LTR（RSV / 5&#39;LTRハイブリッドプロモーター）、Ψ（HIV-1Ψパッケージングシグナル）、RRE（HIV-1のRev応答要素）。 <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="図7" /> 図7。 U2-OSのタイタープレート。U2-OSを6ウェルプレート上に播種し、異なるウイルス濃度（COの1/100と1/10、1、5、10μlの希釈を用いてトランスフェクトした）ウイルス上清をncentrated。一つはよくトランスフェクトしていないコントロール（NT）として使用した。 6-7日後、細胞をクリスタルバイオレットと空気乾燥で染色した。

<ol>
	<li>Coux, O., Tanaka, K., Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+20S+and+26S+proteasomes.">Structure and functions of the 20S and 26S proteasomes.</a> Annu. Rev. Biochem. 65, 801-847 (1996).</li><li>Davies, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+oxidized+proteins+by+the+20S+proteasome.">Degradation of oxidized proteins by the 20S proteasome.</a> Biochimi. 83, 301-310 (2001).</li><li>Stangl, K., Stangl, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+pathway+and+endothelial+(dys)function.">The ubiquitin-proteasome pathway and endothelial (dys)function.</a> Cardiovasc. Res. 85, 281-290 (2009).</li><li>Tuoc, T. C., Stoykova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+ubiquitin-proteosome+system+in+neurogenesis.">Roles of the ubiquitin-proteosome system in neurogenesis.</a> Cell Cycle. 9, 3174-3180 (2010).</li><li>Rock, K. L., 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=Inhibitors+of+the+proteasome+block+the+degradation+of+most+cell+proteins+and+the+generation+of+peptides+presented+on+MHC+class+I+molecules.">Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.</a> Cell. 78, 761-771 (1994).</li><li>Lehman, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+proteasome+system+in+neuropathology.">The ubiquitin proteasome system in neuropathology.</a> Acta Neuropathol. 118, 329-347 (2009).</li><li>Koziel, R., Greussing, R., Maier, A. B., Declercq, L., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Interplay+between+mitochondrial+and+proteasome+activity+in+skin+aging.">Functional Interplay between mitochondrial and proteasome activity in skin aging.</a> J. Invest. Dermatol. 131, 594-603 (2010).</li><li>Grillari, J., Grillari-Voglauer, R., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+cellular+proteins+by+ubiquitin+and+ubiquitin-like+molecules:+role+in+cellular+senescence+and+aging.">Post-translational modification of cellular proteins by ubiquitin and ubiquitin-like molecules: role in cellular senescence and aging.</a> Adv. Exp. Med. Biol. 694, 172-196 (2010).</li><li>Bulteau, A. L., Szweda, L. I., Friguet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+declines+in+proteasome+activity+in+the+heart.">Age-dependent declines in proteasome activity in the heart.</a> Arch. Biochem. Biophys. 397, 298-304 (2002).</li><li>Strucksberg, K. H., Tangavelou, K., Schroder, R., Clemen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasomal+activity+in+skeletal+muscle:+A+matter+of+assay+design,+muscle+type,+and+age.">Proteasomal activity in skeletal muscle: A matter of assay design, muscle type, and age.</a> Anal. Biochem. , (2009).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-lived+green+fluorescent+proteins+for+quantifying+ubiquitin/proteasome-dependent+proteolysis+in+living+cells.">Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.</a> Nat. Biotechnol. 18, 538-543 (2000).</li><li>Lindsten, K., Menendez-Benito, V., Masucci, M. G., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+mouse+model+of+the+ubiquitin/proteasome+system.">A transgenic mouse model of the ubiquitin/proteasome system.</a> Nat. Biotechnol. 21, 897-902 (2003).</li><li>Liu, 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=Impairment+of+the+ubiquitin-proteasome+system+in+desminopathy+mouse+hearts.">Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.</a> FASEB J. 20, 362-364 (2006).</li><li>Bowman, A. B., Yoo, S. Y., Dantuma, N. P., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+dysfunction+in+a+polyglutamine+disease+model+occurs+in+the+absence+of+ubiquitin-proteasome+system+impairment+and+inversely+correlates+with+the+degree+of+nuclear+inclusion+formation.">Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation.</a> Hum. Mol. Genet. 14, 679-691 (2005).</li><li>Myung, J., Kim, K. B., Lindsten, K., Dantuma, N. P., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+proteasome+active+site+allostery+as+revealed+by+subunit-specific+inhibitors.">Lack of proteasome active site allostery as revealed by subunit-specific inhibitors.</a> Mol. Cell. 7, 411-420 (2001).</li><li>Menendez-Benito, V., Heessen, S., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+ubiquitin-dependent+proteolysis+with+green+fluorescent+protein+substrates.">Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.</a> Methods Enzymol. 399, 490-511 (2005).</li><li>Lener, 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+NADPH+oxidase+Nox4+restricts+the+replicative+lifespan+of+human+endothelial+cells.">The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells.</a> Biochem. J. 423, 363-374 (2009).</li></ol>

特別な利害関係は宣言されません。

ユビキチン-プロテアソーム活性のためのレポーター基質として緑色蛍光タンパク質（GFP）を用いた最初の出版は2000年12年に出版された。それ以来、GFPは細胞の活動、特にユビキチン - プロテアソームプロセスを可視化するための共通のツールと​​なっています。 in vivoでのユビキチン-プロテアソーム活性をモニターするために、GFP-ベースのレポータートランスジェニックマ...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>のpEGFP-C1ベクター</td><td> BD Bioscience社クロンテック</td><td> 6084から1 </td></tr><tr><td> pENTR方向性TOPOクローニングキット</td><td>インビトロジェン</td><td> K2400-20 </td></tr><tr><td> pLenti6/V5方向性TOPOクローニングキット</td><td>インビトロジェン</td><td> V496-10 </td></tr><tr><td>リポフェクトアミン2000試薬</td><td>インビトロジェン</td><td> 11668019 </td></tr><tr><td> DMEM </td><td>シグマ</td><td> D5546 </td></tr><tr><td> PVDFフィルター（Rotilabo-Spritzenfilter） </td><td>ロート</td><td> P667.1 </td></tr><tr><td>ポリエチレングリコール</td><td>シグマ</td><td> P2139 </td></tr><tr><td> NaClを</td><td>メルク</td><td> 1.06404.1000 </td></tr><tr><td>ダルベッコのリン酸は、サリのバッファNE 1X（PBS）で</td><td>インビトロジェン</td><td> 14190 </td></tr><tr><td>ヘキサジメトリンブロマイド</td><td>シグマ</td><td> 10,768-9 </td></tr><tr><td>ブラストサイジン</td><td>インビトロジェン</td><td> R21001 </td></tr><tr><td>クリスタルバイオレット</td><td>シグマ</td><td> C3886 </td></tr><tr><td> FACSチューブ</td><td> BD Biosciences社</td><td></td></tr><tr><td>ペニシリンストレプトマイシン（ペンストレプトマイシン） </td><td>インビトロジェン</td><td> 15140130 </td></tr><tr><td> L-グルタミン、200mMの</td><td>インビトロジェン</td><td> 25030024 </td></tr><tr><td>ウシ胎児血清（FBS） </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM非必須アミノ酸（NEAA）100倍</td><td>インビトロジェン</td><td> 11140035 </td></tr><tr><td> MEMピルビン酸ナトリウムを100mM </td><td>インビトロジェン</td><td> 11360039 </td></tr><tr><td> D-（+） - グルコース（45％） </td><td>シグマ</td><td> G8769 </td></tr><tr><td>ジェネティシン</td><td>インビトロジェン</td><td> 11811023 </td></tr><tr><td> CaCl2を</td><td>メルク</td><td> C5080 </td></tr><tr><td>ヘペス</td><td>シグマ</td><td> H3375 </td></tr><tr><td>トリプシン-EDTA（0.05％） </td><td>インビトロジェン</td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

プロテアソームは異常、誤って折り畳まれた、破損したり、酸化されたタンパク質1、2のタンパク質分解に関与する主な細胞内小器官である。プロテアソーム活性の維持は、細胞のストレス応答3、細胞周期の調節と細胞分化4または免疫システムの応答5のように、多くの重要な細胞プロセスに関与していた。ユビキチン-プロテアソーム系の機能不全は、腫瘍および神経変性疾患4、6の開発に関係している。さらに、プロテアソーム活性の低下は、細胞老化と生物の老化7、8、9、10の特徴として見出された。ここで、我々は、GFP-DGN融合タンパク質を用いて、生細胞におけるユビキチン - プロテアソーム活性を測定する方法を提案する。初代培養細胞を生体内でユビキチン - プロテアソーム活性をモニターすることができるように、相補的DNA（緑色蛍光タンパク質（GFP）-DGN融合タンパク質をコードする構築物のGFPDGN、不安定な）とフレームシフト突然変異を運ぶバリアント（GFP-dgnFS、安定した11）レンチウイルス発現ベクターに挿入されます。それは細胞のタイプやドナーの年齢とは無関係非常に高いトランスフェクション効率を保証しますので、我々は伝統的なトランスフェクション技術を介して、この手法を好む。プロテアソーム阻害剤の非存在下または存在下でGFP-dgnFS（安定した）タンパク質と不安定化タンパク質（GFP-DGN）によって表示される蛍光の差は、それぞれの特定の細胞株ではユビキチン - プロテアソーム活性を推定するために使用することができます。これらの違いは、落射蛍光顕微鏡によって監視することができますまたはフローサイトメトリーで測定することができる。

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

生きた細胞内でユビキチン - プロテアソーム活性をモニターするための方法が記載されている。デグロン - 不安定化のGFP（GFP-DGN）と安定したGFP-dgnFS融合タンパク質が生成され、レンチウイルス発現ベクターを用いて細胞内に伝達される。この手法は、ユビキチン - プロテアソーム活性を容易に落射蛍光またはフローサイトメトリーを用いて評価することができる安定したGFP-dgn/GFP-dgnFS発現する細胞株を生成することができます。

デグロンを使用した生細胞におけるユビキチン·プロテアソーム活性のモニタリング（DGN）·不安定化緑色蛍光タンパク質（GFP）ベースのレポータータンパク質

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

16.6K ビュー.  Institute for Biomedical Aging Research. 生きた細胞内でユビキチン - プロテアソーム活性をモニターするための方法が記載されている。デグロン - 不安定化のGFP（GFP-DGN）と安定したGFP-dgnFS融合タンパク質が生成され、レンチウイルス発現ベクターを用いて細胞内に伝達される。この手法は、ユビキチン - プロテアソーム活性を容易に落射蛍光またはフローサイトメトリーを用いて評価すること?...

ビデオ: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

ヘリオス遺伝子銃を用いたタマネギの細胞における蛋白質 - 蛋白質相互作用のための二分子蛍光相補（BiFC）アッセイ

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

胚性幹細胞を生体内でクロマチン蛋白質のダイナミクスを測定するアッセイ（FRAP＆FLIP）を光退色

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

タンパク質分解の体系的な分析のためのレポーターベースの成長アッセイ

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

生細胞内のタンパク質断片相補アッセイ（PCA）によるゲノムワイドなタンパク質間相互作用のスクリーニング

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

ハイスループットシーケンシングを利用し全体プロテイン飽和突然変異誘発ライブラリの機能評価のためのプロトコル

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...