Protease and phosphatase inhibitor cocktail

Western blot was performed as described previously [35 (link)]. GMECs were lysed with ice-cold RIPA buffer (Solarbio, Beijing, China) containing protease and phosphatase inhibitor cocktail (Solarbio, Beijing, China). The protein concentrations were examined with a BCA kit (Epizyme, Shanghai, China). The quantified protein from each group was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Following blocking with 5% skimmed milk in Tris-Buffered Saline and Tween 20 (TBST) buffer for 2 h at room temperature, the membranes were incubated with primary antibodies at 4 ℃ overnight. After washing five times with TBST for 5 min each time, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The blots were washed three times with TBST and determined with a chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA). The levels of target protein bands were quantified with Image J software. The primary antibodies used for Western blot are listed in Table 2.

Snap-frozen colon tissues and harvested cells were lysed in RIPA lysis buffer (R0010, Solarbio) containing protease and phosphatase inhibitor cocktail (P1261, Solarbio). The concentrations of proteins were determined using the BCA Protein Assay Kit (PC0020, Solarbio). The protein samples (20μg/40μg per lane) were separated by 7.5% or 15% SDS polyacrylamide gel and transferred to polyvinylidene fluoride membranes (0.45 μm; Millipore) using a semi-dry transfer method. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2h at room temperature, followed by incubation with primary antibodies against CD73 (1:1000, # 87661SF, CST), ZO-1 (1:1000, A0659, ABclonal), Occludin (1:1000, A12621, ABclonal), Claudin-1 (1:1000, A21971, ABclonal), p-STAT3 (1:2000, #9145S, CST), STAT3(1:1000, #12640S, CST) and β-Actin (1:1000, #4970S, CST) at 4°C overnight. The membranes were washed three times in TBST for 5 min each and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000, #7074S; CST) for 45 min. The target proteins were visualized using an enhanced chemiluminescence substrate kit (WP20005, Thermo Fisher Scientific). The densitometry of protein bands was quantified and analyzed using ImageJ software.

Proteins were extracted from colon tissues of CD patients using radioimmunoprecipitation assay lysis buffer with phenylmethanesulfonylfluoride and a protease and phosphatase inhibitor cocktail (Solarbio, China). After protein concentrations were quantified, they were balanced with SDS-PAGE sample loading buffer (Beyotime). Protein samples were transferred to polyvinylidene fluoride membranes (General Electrics Healthcare, USA), blocked with 5% nonfat milk for 1 hour, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-WTAP (1:1000, Abcam, UK) and rabbit anti-METTL14 (1:1000, Abcam). Mouse anti-β-actin (1:5000, Proteintech) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase were considered as internal references. After washing 3 times with tris-buffered saline containing tween 20, the membranes were incubated with the appropriate horseradish peroxidase-labeled goat secondary antibodies (1:5000, Thermo Fisher Scientific, USA) at room temperature for 1 hour. The membranes were incubated with enhanced chemiluminescence solution (Applygen, China) for 1 minute and visualized using a chemiluminescence imager (Clinx Science Instruments, China).