Name: analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands
Uploaded: 2017-05-30T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

I.P. is funded by a Natural Sciences and Engineering Research Council of Canada grant (NSERC #418233-2012); a Fonds de Recherche du Qu&#233;bec-Sant&#233; (FRQS), a Quebec Breast Cancer Foundation career award, and a Leader Founds grant from the Canadian Foundation for Innovation grant. E.D. received a scholarship from the Fondation universitaire Armand-Frappier.

All animal protocols used in this study were approved by the University Animal Care Committee (INRS-Institut Armand-Frappier, Laval, Canada).
1. Identifying Protein Co-localization
<ol>
	<li>From tissue to microscopic slides 
	NOTE: Tissues and sections should be handled on dry ice.
	<ol>
		<li>Excise the mammary glands from an animal (for a complete description of this procedure, refer to Plante et al.)14.</li>
		<li>Embed the excised t...

<ol>
	<li>Sternlicht, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+stages+in+mammary+gland+development:+the+cues+that+regulate+ductal+branching+morphogenesis.">Key stages in mammary gland development: the cues that regulate ductal branching morphogenesis.</a> Breast Cancer Res. 8 (1), 201 (2006).</li><li>Oakes, S. R., Hilton, H. N., Ormandy, C. 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+alveolar+switch:+coordinating+the+proliferative+cues+and+cell+fate+decisions+that+drive+the+formation+of+lobuloalveoli+from+ductal+epithelium.">The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium.</a> Breast Cancer Res. 8 (2), 207 (2006).</li><li>Stein, T., Hilton, H. N., Ormandy, C. 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+alveolar+switch:+coordinating+the+proliferative+cues+and+cell+fate+decisions+that+drive+the+formation+of+lobuloalveoli+from+ductal+epithelium.">The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium.</a> Breast Cancer Res. 8 (2), 207 (2006).</li><li>El-Sabban, M. E., Abi-Mosleh, L. F., Talhouk, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+gap+junctions+and+their+role+in+mammary+epithelial+cell+differentiation.">Developmental regulation of gap junctions and their role in mammary epithelial cell differentiation.</a> J Mammary Gland Biol Neoplasia. 8 (4), 463-473 (2003).</li><li>Hennighausen, L., Robinson, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+networks+in+the+mammary+gland.">Information networks in the mammary gland.</a> Nat Rev Mol Cell Biol. 6 (9), 715-725 (2005).</li><li>Gudjonsson, T., Adriance, M. C., Sternlicht, M. D., Petersen, O. W., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoepithelial+cells:+their+origin+and+function+in+breast+morphogenesis+and+neoplasia.">Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia.</a> J Mammary Gland Biol Neoplasia. 10 (3), 261-272 (2005).</li><li>Nguyen, D. A., Neville, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+regulation+in+the+mammary+gland.">Tight junction regulation in the mammary gland.</a> J Mammary Gland Biol Neoplasia. 3 (3), 233-246 (1998).</li><li>Stewart, M. K., Simek, J., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+role+of+connexins+in+mammary+gland+morphogenesis+and+function.">Insights into the role of connexins in mammary gland morphogenesis and function.</a> Reproduction. 149 (6), R279-R290 (2015).</li><li>Dianati, E., Poiraud, J., Weber-Ouellette, A., Connexins Plante, I., E-cadherin, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claudin-7+and+beta-catenin+transiently+form+junctional+nexuses+during+the+post-natal+mammary+gland+development.">Claudin-7 and beta-catenin transiently form junctional nexuses during the post-natal mammary gland development.</a> Dev Biol. 416 (1), 52-68 (2016).</li><li>Derangeon, M., Spray, D. C., Bourmeyster, N., Sarrouilhe, D., Herve, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+influence+of+connexins+and+apical+junction+proteins+on+their+expressions+and+functions.">Reciprocal influence of connexins and apical junction proteins on their expressions and functions.</a> Biochim Biophys Acta. 1788 (4), 768-778 (2009).</li><li>Hernandez-Blazquez, F. J., Joazeiro, P. P., Omori, Y., Yamasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+intracellular+movement+of+connexins+by+E-cadherin+in+murine+skin+papilloma+cells.">Control of intracellular movement of connexins by E-cadherin in murine skin papilloma cells.</a> Exp Cell Res. 270 (2), 235-247 (2001).</li><li>Kiyokawa, E., Hara, S., Nakamura, T., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+(Forster)+resonance+energy+transfer+imaging+of+oncogene+activity+in+living+cells.">Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells.</a> Cancer Sci. 97 (1), 8-15 (2006).</li><li>Gustafsdottir, S. 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=Proximity+ligation+assays+for+sensitive+and+specific+protein+analyses.">Proximity ligation assays for sensitive and specific protein analyses.</a> Anal Biochem. 345 (1), 2-9 (2005).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J Vis Exp. (53), (2011).</li><li>Green, M. R., Sambrook, J., Sambrook, 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> Molecular cloning: a laboratory manual. , (2012).</li><li>Taylor, S. C., Berkelman, T., Yadav, G., Hammond, 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+defined+methodology+for+reliable+quantification+of+Western+blot+data.">A defined methodology for reliable quantification of Western blot data.</a> Mol Biotechnol. 55 (3), 217-226 (2013).</li><li>Oxford, E. 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=Molecular+composition+of+the+intercalated+disc+in+a+spontaneous+canine+animal+model+of+arrhythmogenic+right+ventricular+dysplasia/cardiomyopathy.">Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy.</a> Heart Rhythm. 4 (9), 1196-1205 (2007).</li><li>Wang, X., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+mislocalization:+mechanisms,+functions+and+clinical+applications+in+cancer.">Protein mislocalization: mechanisms, functions and clinical applications in cancer.</a> Biochim Biophys Acta. 1846 (1), 13-25 (2014).</li><li>Bertocchi, C., 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=Nanoscale+architecture+of+cadherin-based+cell+adhesions.">Nanoscale architecture of cadherin-based cell adhesions.</a> Nat Cell Biol. 19 (1), 28-37 (2017).</li><li>Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., Chu, 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+Guide+to+Fluorescent+Protein+FRET+Pairs.">A Guide to Fluorescent Protein FRET Pairs.</a> Sensors (Basel). 16 (9), (2016).</li><li>Fredriksson, 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=Proteomic+analysis+of+proteins+surrounding+occludin+and+claudin-4+reveals+their+proximity+to+signaling+and+trafficking+networks.">Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.</a> PLoS One. 10 (3), e0117074 (2015).</li><li>Weibrecht, I., 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=Proximity+ligation+assays:+a+recent+addition+to+the+proteomics+toolbox.">Proximity ligation assays: a recent addition to the proteomics toolbox.</a> Expert Rev Proteomics. 7 (3), 401-409 (2010).</li><li>Malovannaya, 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=Streamlined+analysis+schema+for+high-throughput+identification+of+endogenous+protein+complexes.">Streamlined analysis schema for high-throughput identification of endogenous protein complexes.</a> Proc Natl Acad Sci U S A. 107 (6), 2431-2436 (2010).</li></ol>

Cell-cell interactions via junctions are required for the proper function and development of many organs, such as the mammary gland. Studies have shown that junctional proteins can regulate the function and stability of one another and activate signal transduction by tethering each other at the cell membrane10. The protocols presented in the current manuscript have provided interesting findings about junctional protein differential expression, localization, and interaction during normal murine gla...

Mammary gland growth and development occurs mainly after birth. This organ constantly remodels itself throughout the reproductive life of a mammal1. The adult mammary gland epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of basal cells, mainly composed of myoepithelial cells, surrounded by a basement membrane2. For a good review on mammary gland structure and development, the reader can refer to Sternlicht1. Cell-cell interactions via gap (GJ), tight (TJ), and adherens (AJ) junctions are necessary for the normal development and function of the gland1,3,4,5,6. The main components of these junctions in the murine mammary gland are Cx26, Cx30, Cx32, and Cx43 (GJ); Claudin-1, -3, -4, and -7 and ZO-1 (TJ); and E-cadherin, P-cadherin, and &#946;-catenin (AJ)7,8. The levels of expression of these different junctional proteins vary in a stage-dependent manner during mammary gland development, suggesting differential cell-cell interaction requirements9. GJ, TJ, and AJ are linked structurally and functionally and tether other structural or regulatory proteins to the neighboring sites of adjacent cells, thus creating a junctional nexus10. The composition of the junctional nexus can impact bridging with the underlying cytoskeleton, as well as nexus permeability and stability, and can consequently influence the function of the gland8,9,10,11. The components of intercellular junctions residing in junctional nexuses or interacting with one another at different developmental stages of mammary gland development were analyzed recently using co-immunofluorescence (co-IF) and co-immunoprecipitation (co-IP)9. While other techniques allow for the evaluation of the functional association between proteins, these methods are not presented in this manuscript.
As proteins merely act alone to function, studying protein-protein interactions (PPIs), such as signal transductions and biochemical cascades, is essential to many researchers and can provide significant information about the function of proteins. Co-IF and microscopic analysis help to evaluate a few proteins that share the same subcellular space. However, the number of targets is limited by the antibodies, which must be raised in different animals, and by the access to a confocal microscope equipped with different wavelength lasers and a spectral detector for multiplexing. Co-IP confirms or reveals high-affinity physical interactions between two or more proteins residing within a protein complex. Despite the development of novel techniques, such as fluorescence resonance energy transfer (FRET)12 and proximity ligation assay (PLA)13, which can simultaneously detect the localization and interactions of proteins, co-IP remains an appropriate and affordable technique to study interactions between endogenous proteins.
The step-by-step method described in this manuscript will facilitate the study of protein localization and PPIs and point out pitfalls to avoid when studying endogenous PPIs in the mammary glands. The methodology starts with the presentation of the different preservation procedures for the tissues required for each technique. Part 1 presents how to study protein co-localization in three steps: i) the sectioning of mammary glands, ii) the double- or triple-labeling of different proteins using the co-IF technique, and iii) the imaging of protein localization. Part 2 shows how to precipitate an endogenous protein and identify its interacting proteins in three steps: i) lysate preparation, ii) indirect protein immunoprecipitation, and iii) binding partner identification by SDS-PAGE and Western blot. Each step of this protocol is optimized for rodent mammary gland tissues and generates high-quality, specific, and reproducible results. This protocol can also be used as a starting point for PPI studies in other tissues or cell lines.

<table>
	<tbody>
		<tr>
			<td>Mice strain and stage&#160;</td>
			<td>St. Constant, Quebec, Canada</td>
			<td></td>
			<td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&#160;</td>
		</tr>
		<tr>
			<td>PBS 10X (stock)</td>
			<td></td>
			<td></td>
			<td>1)&#160;&#160;&#160;&#160;&#160; Dissolve 80g NaCl (F.W.: 58.44), 2g KCl (F.W. 74.55), 26.8g Na2HPO4&#8226;7H2O (F.W. 268.07) and 2.4g KH2PO4 (F.W.:136.09) in 800ml distilled water.&#160; 
			2)&#160;&#160;&#160;&#160;&#160; Adjust the PH to 7.4&#160; 
			3)&#160;&#160;&#160;&#160;&#160; Add water to reach to the 1 litre final volume.&#160;</td>
		</tr>
		<tr>
			<td>TBS 10X (stock)</td>
			<td></td>
			<td></td>
			<td>1)&#160;&#160;&#160;&#160;&#160; Dissolve 60.5g TRIS, 87.6g NaCl in 800ml distilled water.&#160; 
			2)&#160;&#160;&#160;&#160;&#160; Adjust the PH to 7.5&#160; 
			3)&#160;&#160;&#160;&#160;&#160; Add water to reach to the 1 litre final volume.&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 1-Immunofluorescence</td>
		</tr>
		<tr>
			<td>Freezing media</td>
			<td>VWR International, Ville Mont-Royal, QC, Canada</td>
			<td>95067-840</td>
			<td>VMR frozen sections compound&#160;</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Mississauga, ON, Canada</td>
			<td>956640</td>
			<td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115V 60Hz</td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>C.L. Sturkey, Inc. Les Produits&#160;Scientifiques&#160;ESBE&#160;St-Laurent, QC, Canada</td>
			<td>&#160;BLM1001C</td>
			<td>High profile gold coated blades</td>
		</tr>
		<tr>
			<td>Pap pen</td>
			<td>Cedarlane, Burlington, ON, Canada</td>
			<td>8899</td>
			<td>Super PAP Pen, Thermo fisher scientific</td>
		</tr>
		<tr>
			<td>Microscopic slides</td>
			<td>Fisher Scientific, Burlington, ON, Canada</td>
			<td>12-550-15</td>
			<td>Fisherbrand&#160;Superfrost Plus Microscope Slides</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>BioShop Canada Inc, Burlington, ON, Canada</td>
			<td>FOR201.1</td>
			<td>Forlmadehyde</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>Santa Cruz Biotechnology, Inc, California, USA</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>3% BSA in TBS</td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td></td>
			<td></td>
			<td>TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Oakville, ON, Canada</td>
			<td>P 9416</td>
			<td>Tween 20, Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Cedarlane, Burlington, ON</td>
			<td>17984-25(EM)</td>
			<td>Fluoromount-G</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&#160;</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Life technologies, Waltham, MA, USA &#38; Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&#160;</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&#160;</td>
			<td>Mentioned in Column D</td>
			<td>&#946;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Life technologies, Waltham, MA, USA &#38; Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>Connexin26&#160; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&#160;</td>
		</tr>
		<tr>
			<td>Nuclei stain</td>
			<td>Fisher Scientific, Burlington, ON, Canada</td>
			<td>D1306</td>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1000 in PBS</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon Canada, Mississauga, On, Canada</td>
			<td></td>
			<td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&#160;</td>
		</tr>
		<tr>
			<td>Software of IF images analysis</td>
			<td>Nikon Canada, Mississauga, On, Canada</td>
			<td></td>
			<td>NIS-elements software (version 4)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 2-Immunoprecipitation</td>
		</tr>
		<tr>
			<td>Triple-detergent Lysis buffer (100ml) pH=8.0&#160;</td>
			<td></td>
			<td></td>
			<td>1) Mix 50mM TRIS (F.W.&#160;:121.14), 150mM NaCl (F.W.&#160;:58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80ml distilled H2O.&#160; 
			2) Adjust the PH to 8.0 with HCl 6N (~0.5ml). 
			3) Adjust the volume to 100ml. Keep it in fridge.&#160; 
			At the day of protein extraction, use 1/100 NaVo3, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer: 
			Sodium Fluorid (stock) solution 1.25M (F.W. 41.98), Sodium Orthovanadate (stock) Solution 1M (F.W.: 183.9)</td>
		</tr>
		<tr>
			<td>Protease/phosphatase inhibitor</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>78441</td>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X)</td>
		</tr>
		<tr>
			<td>Protein dosage</td>
			<td>Thermo Scientific, Rockford, Illinois, USA</td>
			<td>23225</td>
			<td>Pierce BCA protein assay kit&#160;</td>
		</tr>
		<tr>
			<td>Tissue grinder</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>FTH-115</td>
			<td>Power 125, Model FTH-115</td>
		</tr>
		<tr>
			<td>Magnetic beads and stand</td>
			<td>Millipore, Etobicoke, ON, Canada</td>
			<td></td>
			<td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td>
		</tr>
		<tr>
			<td>Wash solution for IP</td>
			<td></td>
			<td></td>
			<td>PBS or PBS-Tween20 0.1% depending to the step</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &#181;l/200 &#181;l</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Sigma-Aldrich, Oakville, ON, Canada</td>
			<td>Mentioned in coulmn D</td>
			<td>Connexin43 (#C6219) (Sigma-Aldrich) 4 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Laemmli buffer&#160;</td>
			<td>BIO-RAD, Mississauga, Ontario, Canada</td>
			<td>1610747</td>
			<td>4x Laemmli Sample Buffer (Add &#946;-mercaptoethanol following manufacturer recommendation)</td>
		</tr>
		<tr>
			<td>Acidic glycine&#160;</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>PB381-5</td>
			<td>0.2 M glycine; adjust pH=2.5 with HCl&#160;</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>BP152-1</td>
			<td>1 M (pH=8)&#160;</td>
		</tr>
		<tr>
			<td>SDS-PAGE acrylamide gels&#160;</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1610180 -5</td>
			<td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td>
		</tr>
		<tr>
			<td>Running buffer 10x&#160;</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704272</td>
			<td>Tris 30.3g/glycine 144.1g /SDS 10g in 1 litre distilled water</td>
		</tr>
		<tr>
			<td>Membranes</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704272</td>
			<td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
		</tr>
		<tr>
			<td>Transfer method</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704155</td>
			<td>Trans-Blot Turbo Transfer System</td>
		</tr>
		<tr>
			<td>Dry Milk</td>
			<td>Smucker Food of Canada Co, Markham, ON, Canada</td>
			<td></td>
			<td>Fat Free Instant Skim Milk Powder, Carnation</td>
		</tr>
		<tr>
			<td>Blocking solution for blots</td>
			<td></td>
			<td></td>
			<td>5% dry milk in TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Washing solutions for blots</td>
			<td></td>
			<td></td>
			<td>TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Sigma-Aldrich, Oakville, Ontario &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Cell Signaling, Beverly, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>E-cadherin (24E10) rabbit mAb 1/1000 (#3195s) (Cell Signaling) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody&#160; (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Life technologies, Waltham, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Claudin-7 (#34-9100) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366)&#160; 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Life technologies, Waltham, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Claudin3&#160; (#34-1700) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody&#160; (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Luminol solution for signal detection on blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1705061&#160;</td>
			<td>Clarity Western ECL Blotting Substrate&#160;</td>
		</tr>
		<tr>
			<td>Imaging blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1708280</td>
			<td>ChemiDoc MP imaging system</td>
		</tr>
		<tr>
			<td>Analayzing blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td></td>
			<td>ImageLab 5.2 software&#160;</td>
		</tr>
	</tbody>
</table>

analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

To determine whether GJ, AJ, and TJ components can interact together in the mammary gland, co-IF assays were first performed. Cx26, a GJ protein, and &#946;-Catenin, an AJ protein, were probed with specific antibodies and revealed using fluorophore-conjugated mouse-647 (green, pseudocolor) and goat-568 (red) antibodies, respectively (Figure 1B and C). Data showed that they co-localize at the cell membrane of epithelial cells in the mice mammary gland on l...

Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

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