Immunoblot Analysis of Cell Signaling

Cell lysates were prepared at 75% of confluence by using 500 μL of radio-immunoprecipitation assay buffer (25 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in 10-cm culture dishes with a 20-minute incubation on ice. The protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Hercules, CA). Immunoblot analyses were performed as previously described [37 (link), 38 (link)]. β-Actin antibodies were obtained from Santa Cruz Biotechnology. HER2, Claudin-1, ZEB1, ZO-1, Fak, pFak, Smad, pSmad were purchased from Cell Signaling Technology. The secondary antibodies were the F(ab)₂ fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The immunoblot reagents were from an electrochemiluminescence kit (Amersham Biosciences, NJ, USA).