Edta free protease inhibitor cocktail

Proteins were extracted using RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with EDTA-free protease inhibitor cocktail (cat. no. 04693159001; Roche Diagnostics GmbH) as previously described (16 (link)). The suspension was collected after centrifugation at 12,000 × g for 15 min at 4°C, followed by protein concentration determination using BCA assay. Equal amounts of proteins (20–40 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membranes using a Bio-Rad semi-dry transfer system. Then the membrane was blocked with 5% skimmed milk. After blocking, the membranes were incubated with primary antibodies (dilution ratio of 1:1,000 with PBST containing 5% BSA) overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies (dilution ratio of 1:5,000 with PBST containing 5% non-fat milk; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The protein bands were visualized using the Tanon 4600 imaging system (Tanon Science and Technology Co., Ltd.) with enhanced chemiluminescence (cat. no. 407207; EMD Millipore; Merck KGaA). The primary antibodies used included Smad7 (cat. no. sc-11392; Santa Cruz Biotechnology, Inc.) and GAPDH (product code ab128915; Abcam).

Western blot analysis was performed as described previously (13 (link)). Briefly, GBC-SD and SGC-996 cells were washed twice with ice-cold PBS and then lysed with ice-cold RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented using EDTA-free protease inhibitor cocktail (cat. no. 11836170001; Roche Diagnostics), and followed by protein concentration determination with BCA assay. Lysates were kept on ice for 30 min and then centrifuged at 14,000 × g for 20 min at 4°C. Equal amounts (20 µg) of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were block with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween-20 for 2 h and then incubated with primary antibodies at 4°C overnight. Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies. The primary antibodies used were: Caspase-8 (cat. no. 9746; Cell Signaling Technology, Inc.), caspase-3 (cat. no. 9662; Cell Signaling Technology, Inc.), cleaved-caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.), eIF4A (cat. no. ab31217; Abcam), c-FLIP (cat. no. ab8421; Abcam) and α-tubulin (cat. no. sc-5286; Santa Cruz Biotechnology, Inc.).