Sc 32233

Cells or fresh tissues were lysed in RIPA lysis buffer with mixture of protease inhibitors (Beyotime #ST506) and PhosSTOP (Roche #04906845001). 30 μg total proteins were subjected to 12% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred to PVDF membranes, which were then blocked with 5% milk for 2 hours. The membranes were then probed with primary antibody for IRE1 (Abcam # ab37073), CHOP (Cell Signaling Technology # 2895), GAPDH (Santa Cruz #sc32233), GRP78 (Abcam # ab32618), p-eIF2α (Cell Signaling Technology # 9721), eIF2α (Cell Signaling Technology # 3597), DUOX1 (Abcam #ab78919), p-PERK (Cell Signaling Technology #3179), PERK (Cell Signaling Technology #3192) at 4°C overnight, and then the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Santa Cruz # sc-2030) for 1.5 hours and finally washed and visualized using Chemiluminescent ECL reagent (Beyotime # P0018).

Western blotting was carried out on lysates of gastric cancer cell line MGC-803 as described previously [21 ] using mouse anti-GAPDH (1:4000, Santa Cruz, SC-32233), Anti-CCT3 (1:200, Abcam, ab174255) rabbit anti-MAP3K7 (1:1000, Abcam, ab109526), rabbit anti-CDC42 (1:1000, Abcam, ab187643), rabbit anti-CDK2 antibodies (1:1000, CST, #2546), mouse anti-CDK6 (1:1000, CST, #3136) and mouse anti-CCND3 (1:1000, CST, #2936). The secondary antibody was an HRP-conjugated anti-rabbit IgG antibody (1:5000, Santa Cruz, sc-2004) or anti-mouse IgG (1:5000, Santa Cruz, sc-2005). The enhanced chemiluminescence reagent (Cat. 32106, ThermoFisher, USA) was used for signal detection.

After infection for 5 days, HCT116 cells were washed with ice-cold PBS and then lysed in 2× SDS sample buffer [100 mM Tris–HCl (pH 6.8), 10 mM EDTA, 4% (w/v) SDS, 10% (v/v) glycine] for 1 h at 4 °C. The lysates were clarified by centrifugation at 13 000× g for 30 min at 4 °C and the supernatants were employed for further analysis. The total protein concentration was estimated using BCA (bicinchoninic acid) protein assay kit. Protein samples (30 μg) were loaded and electrophoresed in a SDS–PAGE (10% gel) at 50 V for 3 h, and subsequently transferred to PVDF membranes (Millipore) at 300 mA for 1.5 h. After being blocked with TBST (Tris-buffered saline Tween-20) [20 mM Tris (pH7.6), 150 mM NaCl, 0.01% Tween-20] containing 5% (w/v) non-fat dried skimmed milk powder for 1 h at room temperature, membranes were probed with rabbit anti-EIF3D (abcam, #ab155419, dilution 1:1000), or mouse anti-GAPDH (Santa cruz, #Sc-32233, dilution 1:60 000) overnight at 4 °C. After washing by TBST, the membrane was incubated with HRP (horseradish peroxidase)-labelled anti-rabbit (Santa cruz, #Sc-2054, dilution 1:5000) or anti-mouse (Santa cruz, #Sc-2005, dilution 1:5000) secondary antibody at room temperature for 2 h. The membranes were analysed using super ECL detection reagent. Each experiment was repeated three times.