Ab32124

Western blotting was performed as per the previously published procedure [26 (link)]. Total protein from the cells was lysed using RIPA buffer (Santa Cruz Biotechnology, USA). Approximately 20 μg of protein extract was loaded onto 12% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were then exposed to 5% skim milk and probed with primary antibodies against LAMC2 (1:1000; ab274376; Abcam), B-cell lymphoma (Bcl)-2 (1:1000; ab32124), Bcl-2-associated X (Bax) (1:1000; ab32503), and GAPDH (1:1000; #5174; Cell Signaling Technology Inc., USA) at 4°C overnight, followed by examination with the corresponding horseradish-peroxidase-conjugated secondary antibodies (1:1000; SA205; Solarbio, China) at 37°C for 1 h. ECL kit (Najm Biotech ECL, Iran) was used to treat the membranes.

Total protein was isolated using a radioimmunoprecipitation assay lysis buffer. The extracted proteins were subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% milk and incubated with the primary antibodies at 4 °C overnight, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence detection kit (Cell Signaling Technology, Danvers, MA, USA). The primary antibodies used were antibodies against Bach1 (1:1500, ab180853), CD44 (1:1500, ab189524), Sox2 (1:1500, ab92494), Nanog (1:1500, ab109250), Oct4 (1:1500, ab181557), p53 (1:1500, ab26), BCL2 (1:1500, ab32124), BAX (1:1500, ab32503), p-p38 MAPK and total p38 MAPK (Cell Signaling Technology, Boston, MA, USA), p-AKT1 (1:1500, phospho S473, ab81283), AKT1 (1:1500, ab28422), c-Fos (1:1500, ab190289), c-Jun (1:1500, ab32137 and GAPDH (1:1500, ab8245),all of which were obtained from Abcam.

Total protein was isolated using RIPA lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. The membrane was and probed with diluted primary rabbit antibodies against Caspase-3 (ab13847), Bcl-2-associated X protein (Bax; ab32503), B-cell lymphoma 2 (Bcl-2) (ab32124), Bcl-XL (ab32370), TGF-β1 (ab92486), β-actin (ab8227), GAPDH (ab181602), E-cadherin (3195), N-cadherin (13116), matrix metalloproteinase (MMP)3 (14351), MMP9 (13667), Smad2 (5339), Smad3 (9523), phosphorylated (p)-Smad2 (18338), and p-Smad3 (9520, Cell Signaling Technologies) overnight at 4°C. The antibodies were from Abcam except for p-Smad2/3. After washing, membrane was re-probed with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205719, 1: 2000, Abcam) for 1 h. The protein bands were visualized using enhanced chemiluminescence (EMD Millipore). β-actin and GAPDH were used as internal controls. The gray values were analyzed with Image J software.