Anti e cadherin rabbit monoclonal antibody

Cell lysates were harvested using RIPA lysis buffer for 30 mins on ice and centrifuged at 13,000 rpm for 10 mins at 4°C. Protein concentration of the supernatant was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., USA). An equal amount of each protein extract (30 μg) was resolved using 10% polyacrylamide gel and electro-transferred onto 0.45 μm hybridization nitrocellulose filter (HATF) membrane (Millipore, USA) using Trans-blot Turbo (Bio-Rad Laboratories, Inc., USA). Membranes were immunoblotted with either rabbit polyclonal anti-IFITM1 antibody (GeneTex, USA), rabbit polyclonal anti-actin antibody (Abcam, USA), rabbit monoclonal anti-SNAIL antibody (Cell Signaling, USA), rabbit monoclonal anti-E-Cadherin antibody (Cell Signaling, USA), mouse monoclonal anti-Fibronectin antibody (Abcam, USA), or mouse monoclonal anti-N-Cadherine antibody (BD, USA) overnight at 4°C. Membranes were incubated with either HRP-conjugated anti-rabbit immunoglobulin (Cell Signaling, USA) or HRP-linked anti-mouse immunoglobulin (Cell Signaling, USA) for 1 hr at room temperature. The protein signal was detected by enhanced chemiluminescence (Thermo, USA) using the Amersham Imager 600 (GE Healthcare Life Sciences, UK).

Protein extracts (cleared lysates) were obtained from cells grown to 70% confluence using standard RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (Quartett GmbH, Berlin, Germany) and 1 mM sodium orthovanadate (Na₃V0₄; Sigma). Cells were collected using a cell scraper, incubated on ice for 5-10 min and centrifuged at 12,000 x g for 20 min at 4°C. Proteins (40 μg) were separated by 10% SDS-PAGE, transferred onto nitrocellulose membranes [7 (link)], blocked with 5% BSA in TBS-Tween for 1 h and incubated overnight at 4°C with the following primary antibodies diluted in 2% BSA: rabbit polyclonal anti-Calretinin (1:10,000; CR 7699/4), rabbit polyclonal anti-FAK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-p-FAK Tyr³⁹⁷ (1:1,000; Cell Signaling Technology), and rabbit polyclonal anti-GAPDH (1:5,000; Sigma); followed by incubation with secondary goat anti-rabbit or anti-mouse (HRP)-labeled antibodies (Sigma) at a dilution of 1:10,000. The signals were detected as described in [7 (link)]. For the EMT analysis membranes were incubated overnight at 4°C with a rabbit monoclonal anti-E-cadherin antibody (1:1,000; #3195; Cell Signaling Technology) and a mouse monoclonal N-cadherin (1:1,000; # 610920; BD Bioscience).

Antibodies were obtained from the following sources: rabbit anti-WNT4 polyclonal antibody (ab91226), rabbit anti-AXIN2 monoclonal antibody (ab109307), rabbit anti-fibronectin polyclonal antibody (ab2413) and rabbit anti-β-catenin monoclonal antibody (ab32572) were obtained from Abcam (Cambridge, MA, USA); rabbit anti-ZO-1 monoclonal antibody (#13663) and rabbit anti-E-cadherin monoclonal antibody (#3195) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (#31460, #31430) were obtained from Invitrogen (Carlsbad, CA, USA). Anti-fluorescein isothiocyanate labeled phalloidin (P5282) and anti-α-SMA mouse monoclonal (A5228) antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Human WNT4 Protein (6076-WN-005/CF) was obtained from R&D Systems (Minneapolis, MN, USA).