Hpa005456

Isolated gastric corpus epithelial cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, and 0.25% sodium deoxycholate) supplemented with 1 mM PMSF and protease inhibitor cocktail (Roche). The concentration of the isolated protein was determined using a detergent-compatible protein assay (Bio-Rad Laboratories). The samples were prepared by boiling in SDS loading buffer (100 mM Tris-HCl, pH 6.8, containing 4% SDS, 0.02% bromophenol blue, and 2% 2-mercaptoethanol). Whole-cell lysates were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The blotted membrane was blocked for 1 h in Tris-buffered saline with Tween 20 (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) containing 5% skim milk and then incubated with the primary antibodies against the ARID1A (Sigma-Aldrich; HPA005456) or GAPDH (Abcam; ab181602). Secondary peroxidase-labeled anti-mouse or anti-rabbit IgG antibodies were purchased from Thermo Fisher Scientific. Membranes were washed with Tris-buffered saline with Tween 20 and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific; 34080) using the ChemiDoc MP system (Bio-Rad Laboratories).