Anti e cadherin rabbit mab

To detect αSMA, RLE cells were washed with ice-cold PBS and lysed with the SDS sample buffer. For the analysis of signaling proteins, washed cells were lysed with a lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH7.5, 100 mM NaCl, 5% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 10 mM Na₃VO₄, and 10 mM NaF). The lysates were cleared by centrifugation and treated with the SDS sample buffer. The samples were subjected to SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting as described previously [49 (link)]. Primary antibodies used were anti-β-tubulin mouse monoclonal antibody (mAb) E7 (Developmental Studies Hybridoma Bank, DSHB), anti-αSMA mouse mAb 1A4 (Sigma), anti-E-cadherin rabbit mAb (Cell Signaling Technology), anti-MEK1/2 rabbit polyclonal antibody (pAb), anti-phospho-MEK1/2 (Ser217/221) rabbit pAb, anti-ERK1/2 rabbit pAb, anti-phospho-ERK1/2 (Thr202/Tyr204) mouse mAb (Cell Signaling Technology), anti-A-Raf rabbit pAb (Santa Cruz Biotechnology), and anti-DA-Raf rabbit pAb [28 (link)]. The blotting bands were detected with a ChemiDoc MP system (Bio-Rad). The band intensity was densitometrically analyzed by using ImageJ software (NIH).

Cells were lysed in sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride (PVDF) membrane. The primary antibodies used included: anti-Akt rabbit mAb (Cat# 4685S, Cell Signaling Technolog, USA), anti-phospho Akt rabbit mAb (Cat# 4060S,Cell Signaling Technology, USA), anti-Vimentin rabbit mAb (Cat# 5741S, Cell Signaling Technology, USA), anti-E-Cadherin rabbit mAb (Cat# 3195,Cell Signaling Technology, USA), anti-PTEN rabbit mAb (Cat# 9188S,Cell Signaling Technology, USA), and anti-p53 rabbit mAb (Cat# 2527S, Cell Signaling Technology, USA). The secondary horseradish peroxidase-conjugated antibody used was HRP-Goat Anti-Rat IgG (H+L) (Cat# SA00001-2, ProteintechGroup, USA). Bands were detected by enhanced chemiluminescence (Amersham, Bucks, UK). Densitometric values were normalized to GAPDH levels.