Bs 10900r

Total cell lysates were lysed in lysis buffer (Beyotime) containing PMSF and an inhibitor cocktail on ice. Protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Aliquots containing 30 µg protein separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat milk for 1 h at room temperature. Membranes were incubated with the corresponding primary antibody (ARHGAP10 at the dilution of 1:2000 and GAPDH at the dilution of 1:5000) at 4 °C overnight, washed with 0.1% Tween20 in TBS, and then incubated with secondary antibody. Primary rabbit anti-ARHGAP10 and anti-GAPDH were purchased from Proteintech Group (55139-1-AP; Wuhan, China) and BIOSS (bs-10900R, China), respectively. The secondary antibody HRP-conjugated goat anti-rabbit IgG was purchased from Abbkine (A21020, China).

The total protein was extracted from transfected GC cells using RIPA buffer (Thermo Fisher Scientific). A BCA protein quantification kit (Beyotime, China) was used to determine the protein concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for protein separation. The gels were transferred to poly (polyvinylidene fluoride) (PVDF) membranes and blocked with 5% nonfat milk for 1 h at room temperature. These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase‑conjugated goat anti‑rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C. Finally, the protein bands were visualized using enhanced chemiluminescence (Pierce, Thermo Fisher Scientific, USA). The grayscale of the protein bands was analyzed using ImageJ software.

For Western blot experiments, 1 × 10⁶ cells were seeded into six-well plates. The coculture and treatment were the same as described above. Then the total proteins were extracted with RIPA buffer containing 1% PMSF and detected with Brasford assay. Proteins of different sizes were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Afterward, proteins were transferred onto a 0.22-μm PVDF membrane and incubated with Tris buffer containing 5% nonfat milk, which was further incubated with primary antibodies, including E-cadherin (#bs-1519R, 1:1,000, Bioss) and β-catenin (#bs-1165R, 1:1,000, Bioss), and secondary antibody (#bs-40295G-HRP, 1:5,000, Bioss) for 12 and 1 h, respectively. Subsequently, the expression of proteins was analyzed using the chemiluminescence analysis system and ImageJ 1.43 software. GAPDH (#bs-10900R, 1:5,000, Bioss) was used as a control for whole-cell lysates. All experiments were performed in triplicate.