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...

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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 ...

The overall goal of this method is to determine whether two or more intercellular junction proteins co-localize within cells and to further assess their physical interaction via co-immunoprecipitation. This method can help to answer key questions in the biology of the mammary gland field including the roles played by junctions in normal development and diseases such as breast cancer. The main advantage of this technique is that it can provide significant information about protein-protein interaction in vivo at a relatively low cost.To perform co-immuno fluorescent staining, retrieve the appropriate microscopic slides from the freezer. Immediately fix the sections by immersing them in four percent formaldehyde for 15 minutes at room temperature. Then, immerse the slides in phosphate-buffered saline at room temperature.Leave the slides there until proceeding to the next step. Circle each section of the slide using a commercially-available hydrophobic barrier or a water-repellent lab pen. Immediately add drops of PBS to the tissue, and place the slide in a human histology chamber for the remainder of the procedure.Block each tissue section with 100 to 200 microliters of three percent Bovine Serum Albumin-Tris-Buffered Saline, 0.1%polysorbate-20, for 30 minutes at room temperature. While the samples are blocking, prepare the primary and secondary antibody solutions by diluting the antibodies in TBS 0.1%polysorbate-20. After removing the blocking solution by aspiration, incubate the sections in 100 to 200 microliters of the diluted primary antibody for 60 minutes at room temperature.Then remove the primary antibody solution by aspiration and wash the sections with 250 to 500 microliters of TBS 0.1%polysorbate-20 for five minutes. Remove the wash solution by aspiration before repeating the wash twice. Aspirate the wash solution and incubate the sections with 100 to 200 microliters of the appropriate Fluoro-4 conjugated secondary antibody for 60 minutes at room temperature.After removing the secondary antibody solution by aspiration, wash the sections with 250 to 500 microliters of TBS 0.1%polysorbate-20 for five minutes. Remove the wash solution and repeat twice. Repeat these steps using the appropriate combination of primary and secondary bodies for any subsequent proteins of interest.Following removal of the final wash, perform the nuclei staining by incubating the section with 100 to 200 microliters of one milligram per millileter DAPI and TBS 0.1%polysorbate-20 for five minutes at room temperature. Aspirate the DAPI solution and mount the slides using a water-soluble, non-fluorescing mounting medium and cover slips. Place the slides flat in a four-degree Celsius refrigerator for at least eight hours.Proceed to the fluorescence microscopic imaging as described in the text protocol. Collect 500 to 1000 micrograms of the lysate and dilute it in PBS to each a final volume of 200 microliters in each 1.5-milliliter tube. Add the antibody against the antigen of interest to the first tube of lysate, and keep it on ice.Incubate the tubes overnight at four degrees Celsius on a tube-roller mixer at low speed. The following day, add 50 microliters of magnetic beads to new 1.5-milliliter tubes for pre-washing. Place the tubes containing the beads on the magnetic stand and allow the beads to migrate towards the magnet.Remove the storage buffer from the beads using a 200-microliter pipette. Wash the beads by adding 500 microliters of PBS 0.1%polysorbate-20, and vortex the tubes vigorously for 10 seconds. Then, put the tubes back onto the magnetic stand and allow the beads to migrate towards the magnet.Remove the excess wash buffer by pipetting with a 200-microliter pipette. Add the lysate antibody reaction complex to the beads and incubate for 90 minutes at room temperature on the roller mixer. Following the incubation, place the tubes on the magnetic stand and allow the beads to migrate toward the magnet.Using a 200-microliter pipette, aspirate and discard the lysate and place the tubes on ice. Wash the beads twice by adding 500 microliters of PBS, placing the tubes on the magnetic stand, and removing the liquid as before. During the wash steps, avoid vortexing, and keep the samples on ice.Wash the beads once with PBS 0.1%polysorbate-20 without vortexing and discard the last wash buffer using a 200-microliter pipette tip. To elute, add 20 microliters of 0.2 molar acidic glycine to the tubes and shake them for seven minutes on the roller mixer. After centrifuging at high speed for a few seconds, collect the supernatant in a fresh, ice-cold tube.Repeat the wash and elution steps for each tube. Then, add 10 microliters of 4x buffer to the 40-microliter eluted sample. Immediately add one molar Tris to the eluted samples, one drop at a time, until its color turns blue, and then proceed to the next tubes.Boil the eluted samples at 70 to 90 degrees Celsius for 10 minutes. Load the prepared lysates and the precipitated samples side-by-side in an SDS-PAGE acrylamide gel. Run the gel in running buffer at 100 volts for approximately 95 minutes, or until the edge of the migrating proteins reaches the bottom of the gel.Subsequently, transfer the gels to a nitrocellulose or PVDF membrane using a standard protocol. Then, block the membrane for one hour on a rocker on low speed in five-percent dry milk TTBS. To identify whether the precipitation was successful, probe the membrane using the first antibody against the precipitated protein.Dilute it in five percent dry milk TTBS at the concentration recommended by the manufacturer. Incubate the membrane overnight at four degrees Celsius on a rocking platform with slow agitation. The following day, wash the membrane three times for five minutes each with TTBS on a rocking platform with high agitation.Then, incubate the membrane in the appropriate secondary antibody conjugated with horseradish peroxidase, diluted in TTBS, for one hour at room temperature on a rocking platform with slow agitation. Perform three to six washes, each for five minutes, with TTBS on a rocking platform with high agitation. Analyze the signal of the secondary antibody by incubating the membrane with a commercially-available luminol solution and follow the manufacturer's instructions.Detect the signal using a chemiluminescence imaging system. To identify interacting proteins, repeat these steps using the appropriate antibodies on the same blot. To determine whether gap junction, adherence junction, and tight junction components can interact together in the mammary gland, co-immunofluorescence assays were performed.The co-immunofluorescence assay shows that beta-catenin, an integral protein of adherens junction, and Connexin 26, a gap junction protein, co-localize at the cell membrane in mice mammary glands on lactation day seven, as reflected by the yellow color. Cell nuclei are visualized with blue DAPI staining. Cryosections from mammary glands on pregnancy day 18 were cut and immunofluorescently stained for Claudin 7, a tight junction protein shown in green, E-Cadherin, an adherens junction protein shown in red, and Connexin 26, a gap junction protein shown in coral blue.Nuclei were stained with blue DAPI. E-Cadherin and Claudin 7 colocalization is displayed as a yellow to light orange color, while Connexin 26 colocalization with E-Cadherin and Claudin 7 resulted in white-punctuated staining. To find out which junctional proteins intermingle and physically tether together at the cell membrane, co-immunoprecipitation was performed using mammary gland tissues from lactating mice.Results showed that Connexin 43, a component of the gap junction, interacts with E-Cadherin and Claudin 7, but not with Claudin 3. These results were confirmed by the reciprocal immunoprecipitation. When E-Cadherin was immunoprecipitated, it interacted with Connexin 43 and Claudin 7.Once mastered, this technique can be done in two days for co-immunofluorescence for four to eight slides, and slides can be imaged the following day if it is performed properly. While attempting the co-immunofluorescence procedure, it is important to remember to choose different Fluoro 4 conjugated antibodies raised in different spaces with non-overlapping wavelengths and to select the proper protein A or G magnetic beads compatible with the antibodies used for co-immunoprecipitation. After its development, this technique paved the way for researchers in the field of cell biology to explore intercellular junctions'interplay in murine mammary glands.Don't forget that working is extremely hazardous and precautions should be always taken while performing this procedure.

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