P-cadherin Monoclonal Antibody (6A9)

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	-Mouse (1:5000)

Protocol tips
-Mouse (1:5000)

Publication protocol

Frozen tissues were ground in liquid nitrogen. The resulting powder was resuspended on dry ice in ice-cold triple detergent lysis buffer (pH: 8) (Tris 50 mM, NaCl 150 mM, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P40, 0.5% sodium deoxycholate) that was supplemented with NaF 1.25 M, NaVO3 1 M and Halt Protease and Phosphatase Cocktail Inhibitor (Fisher Scientific). The homogenates were then incubated for 30 min with slow agitation at 4 °C, followed by sonication. Homogenates were centrifuged at ≥13000 g for 10 min at 4 °C, and the supernatants were aliquoted and stored at −80 °C until later usage. Protein content was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, Illinois, USA). To perform semi-quantitative western blot analysis, between 10 and 50 µg of total protein (Taylor et al., 2013) was loaded onto TGX Stain-Free™ Acrylamide gels (BIO-RAD, Mississauga, Ontario, Canada). Immediately after electrophoresis, the gels were transferred onto PVDF membranes (Trans-blot turbo RTA Transfer kit, BIO-RAD) using the Trans-Blot Turbo Transfer System (BIO-RAD). Total lane proteins were visualized using the ChemiDoc MP imaging system (BIO-RAD) and quantified using ImageLab 5.2 software (BIO-RAD) for normalization. Membranes were blocked with 5% dry milk or 3% BSA in TBS-Tween 0.1% and probed overnight with one of the following primary antibodies: β-catenin (D10A8) rabbit mAb 1/1000 (#8480s), Claudin-1 (#4933s) 1/1000, E-cadherin (24E10) rabbit mAb 1/1000 (#3195s), phosphPlus Stat3 (Tyr 705) (#9130), phospho-Stat5 (Tyr 694) rabbit mAb (#4322s), or Stat5 rabbit Ab (#9363s) from Cell Signaling (Beverly, MA, USA); Claudin-3 (#34–1700) 1/1000, Claudin-4 (#36–4800) 1/1000, Claudin-7 (#34–9100) 1/1000, Connexin-26 (#33–5800) 1/500, Connexin-30 (#71–2200) 1/500 from Life Technologies/Invitrogen (Waltham, MA, USA); Connexin32 (#265-279) 1/1000 or Connexin43 (#C6219) 1/1000 from Sigma-Aldrich (Oakville, Ontario); and P-cadherin 6A9 (#MA1-2003) 1/5000 from Thermo Scientific (Mississauga, ON, Canada). Membranes were washed with TBS-Tween 0.1% and probed with 2nd antibodies (anti-rabbit IgG HRP-linked antibody (#7074s) or anti-mouse IgG HRP-linked antibody (#7076s) (Cell Signaling). The signal was obtained using Clarity™ Western ECL Blotting Substrate (BIO-RAD) and visualized using the ChemiDoc MP imaging system (BIO-RAD). The density of each band was normalized to the total lane proteins using ImageLab 5.2 software.

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