Anti occludin 711500

Affinity-purified polyclonal rabbit anti-EpCAM antibody (Ab) has been described previously [22 (link)]. Monoclonal anti-mouse TROP2 antibody was generated by immunizing rabbits with recombinant protein comprised of the extracellular region of mouse TROP2 fused by human IgG Fc. Polyclonal anti-TROP2, polyclonal goat anti-human HAI-1 (AF1048), sheep anti-matriptase (AF3946), goat anti-mouse HAI-1 (AF1141), and goat anti-mouse HAI-2 (AF1107) Abs were purchased from R & D Systems (Minneapolis, MN, USA). Anti-EpCAM (PA5-19832), anti-claudin-1 (717800), anti-claudin-7 (349100), and anti-occludin (711500) Abs were from Thermo Fisher Scientific (Carlsbad, CA, USA). Rabbit anti-matriptase Ab (IM1014) was from EMD Millipore (Temecula, CA, USA). Polyclonal anti-HAI-2 Ab (HPA011101), mouse anti-Flag mAb (clone M2) and anti-β-actin mAb (clone AC-15) were from Sigma (St. Louis, MO, USA). Anti-E-cadherin mAb was from BD Biosciences (San Jose, CA, USA), and rat anti-HA mAb (clone 3F10) was from Roche (Indianapolis, IN, USA).

Cells were collected and lysed in 1% Triton X-100, 1% NP-40 in PBS, pH 7.4, containing a cocktail of inhibitors of proteases (Roche Applied Science, Mannheim, Germany) and of phosphatases (Sigma-Aldrich). After supernatant recovery, the protein concentration was assessed using the Bio-Rad protein assay kit (Bio-Rad), according to the manufacturer’s protocol. Identical amounts of protein were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Amersham). Membranes were blocked with 5% fat-free milk for 1 h, at room temperature, incubated overnight with primary antibodies at 4°C, and incubated with secondary antibodies for 1 h, at room temperature. Antibodies were as follows: anti-AF6 (610732) and anti-β-Catenin (610153) were from BD Transduction Laboratories^TM; anti-Vimentin (5741), anti-N-Cadherin (13116), anti-Snail (3879), anti-Slug (9585), anti-TCF8/ZEB1 (3396), and anti-E-Cadherin (3195) were from Cell Signaling Technology; anti-GAPDH (sc-47724) was from Santa Cruz Biotechnology; anti-E-cad HECD1 (13-1700), anti-ZO-1 (61-7300), and anti-Occludin (71-1500) were from Thermo Fisher Scientific; anti-αTubulin (T9026) was from Sigma-Aldrich; and horseradish peroxidase-conjugated secondary anti-rabbit (NA934) and anti-mouse (NA931) were from GE Healthcare Life Sciences.