Anti n cadherin

Cell protein was separation with SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked for 2–3 h with 5% non-fat dried milk at room temperature, and then incubated with primary antibody (anti-CD73 mAb was purchased from BBI Life sciences; mAb of anti-E-Cadherin, anti-N-cadherin, anti-Flbronectin1were purchased from R&D Systems; anti-Vimentin mAb were purchased from Abcam) overnight at 4°C. After washed with TBST for three times (15 min each time), membranes were incubated with secondary antibody for 2 h. Proteins were detected with western blotting luminol reagent (Bio-Rad), β-actin or GAPDH was used as the internal standard.

Pyrrole, poly(sodium 4-styrenesulfonate) (PSS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), streptavidin, and sodium hydroxide (NaOH) were obtained from Sigma Aldrich (St. Louis, MO, USA). Anodized aluminum oxide (AAO) membrane filter (pore diameter, 200 nm) was purchased from Whatman (Pittsburgh, PA, USA). Silver paste (P-100 type) was obtained from CANS. NHS-SS-biotin was purchased from CovaChem (Loves Park, Illinois, USA). Biotinylated anti-EpCAM, anti-EGFR, anti-vimentin, anti-N-cadherin, and anti-Trop2 were obtained from R&D system (Minneapolis, MN, USA). Biotinylated anti-CD63 and anti-CD81 were purchased from AnCell (Oak Park, Minnesota, USA). Biotinylated anti-CD9 was obtained from Abcam (Cambridge, UK).

An equal amount of proteins from whole-cell lysates were separated by 4–12% SDS-PAGE and were transferred to a nitrocellulose membrane. Blots were blocked for 1 h with 5% non-fat dry milk and then incubated over night with the following primary antibodies: anti-CA-IX (R&D), anti-N-cadherin, anti-β-catenin and anti-Vimentin (CST-9782), anti-pro-Caspase-3, anti-Cleaved-Caspase-3, anti-AKT (CST-9272), anti-pAKT (CST-9271), anti-ERK (CST-9102), anti-pERK (CST-9101), anti-MMP-2 (CST-33437), anti-CD44 (Abcam), anti-GADPH (G8795) and anti-Actin (A4700). After washing with 0.1% Tween-20 in PBS, the filters were incubated with their respective secondary antibodies for 1 h and analyzed using the ECL system. Densitometric analyses were performed on at least two different expositions to assure the linearity of each acquisition using ImageJ software (v1.46r) [23 (link)].