Ab106066

Total protein was extracted from tissues and cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined by the BCA assay kit (Beyotime, Haimen, Jiangsu, P.R. China). The same amount of protein (50 μg) was separated using 10% SDS-PAGE gel, and then the protein was transfected to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with fat-free milk and incubated with antibody against GATA6 (1:1,000; ab106066; Abcam, Cambridge, MA, USA) and GAPDH (1:1,000; ab8245; Abcam) at 4°C overnight. Finally, the membrane was incubated with HRP-conjugated rabbit anti-mouse IgG (1:5,000; ab6728; Abcam). The protein was detected with the enhanced chemiluminescence method (Beyotime). Protein signals were quantified using Quantity One Software (Bio-Rad, Hercules, CA, USA).

Total protein was extracted from the tissues, and cell lines were cultured using RIPA lysis buffer (Beyotime). The protein concentration was measured by BCA Assay Kit (Beyotime). The same quantity of protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were blocked at room temperature for 1 h with 5% skim milk. The membranes were then incubated with primary antibodies against GATA6 (ab106066) or GAPDH (ab8245) (both from Abcam, Cambridge, MA, USA) at 4°C overnight. Then the membranes were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (ab6728) at room temperature for 1 h. Proteins were detected by ECL Plus Western Blotting Detection Kit (Beyotime). The bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).