Pab30040

Total proteins were extracted from GES-1 cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were cultured for 1 h with primary antibodies against BCL-2 associated X protein (BaX) (1 : 1000, Bioswamp, PAB30040), B-cell lymphoma-2 (Bcl-2) (1 : 1000, Bioswamp, PAB33482), cleaved caspase 3 (1 : 1000, Abcam, Ab2302), density-regulated protein 1 (DRP1) (1 : 1000, Bioswamp, PAB33409) phosphorylation (p)-DRP1 (1 : 1000, Abcam, ab193216), mitochondrial profusion protein Mitofusins (Mfn2) (1 : 1000, Bioswamp, PAB41825), occludin (1 : 1000, Bioswamp, PAB33418), claudin-1 (1 : 1000, Bioswamp, PAB33267), zona occludens 1(ZO-1) (1 : 1000, Bioswamp, PAB36669), phosphatidylinositol-3 kinase (PI3K) (1 : 1000, Bioswamp, PAB30084), p-PI3K (1 : 1000, Abcam, Ab182651), AKT (1 : 1000, Bioswamp, PAB34089), p-AKT (1 : 1000, Abcam, Ab38449), and GAPDH (1 : 1000, Housekeeping, Bioswamp, PAB36269), followed by 1 h of incubation with goat anti-rabbit IgG secondary antibody (1 : 20000, Bioswamp, SAB43714).

Total protein content was extracted from 3 × 105 cells in each group using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) following the manufacturer's protocol. 20 μg of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against CDC2 (Bioswamp, PAB30052, 1 : 1,000), B-cell lymphoma-2 (Bcl-2, Bioswamp, PAB30042, 1 : 1,000), Bcl-2-associated X (Bax, Bioswamp, PAB30040, 1 : 1,000), nuclear factor erythroid-2-related factor 2 (Nrf2, Bioswamp, PAB30175, 1 : 1,000), TrxR1 (abcam, ab124954, 1 : 5,000), apoptosis signal regulating kinase 1 (ASK1, Bioswamp, PAB36297-P, 1 : 1,000), phosphorylated (p)-ASK1 (abcam, ab47304, 1 : 1,000), and GAPDH (Bioswamp, PAB36269, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-labeled goat antirabbit IgG secondary antibody (Bioswamp, PAB160011, 1 : 20,000) for 1 h at room temperature and visualized using a Tanon-5200 apparatus (Tanon, Shanghai, China). The band gray values were read using the TANON GIS software (Tanon). GAPDH acted as the internal reference.