Ab124265

HeLa cells were seeded onto 20 mm glass-bottom cell culture dishes (NEST Scientific) at a density of 0.6×10⁵ cells/ml 24 h before transfection with wild-type GATA4-pcDNA3.1 or mutant GATA4-pcDNA3.1 expression plasmids using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). HeLa cells were fixed with 3.7% formaldehyde/PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100/PBS for 1 h at room temperature 48 h after transfection. Subsequently, cells were incubated with a primary antibody against GATA4 (dilution 1:800; cat. no. ab124265; Abcam) overnight at 4°C and then detected using anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (dilution: 1:500, cat. no. ab150080; Abcam) at room temperature for 2 h. Nuclear staining was performed with a 1:1,000 dilution of DAPI at room temperature for 20 min. The cells were observed under a Leica TCS SP8 Laser Scanning Confocal microscope (Leica Microsystems GmbH).

Cells were lysed in radioimmunoprecipitation assay buffer (89900, Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (78442, Thermo Fisher Scientific) for 30 min on ice, followed by centrifugation at 15,000g for 15 min at 4°C. Proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% bovine serum albumin and probed with antibodies against GATA4 (1:4000; ab124265, Abcam), p21(1:1000; 2947S, Cell Signaling Technology), or β-actin (1:4000; A1978, Sigma-Aldrich). Detection was performed with a chemiluminescent substrate (32132, Thermo Fisher Scientific) followed by exposure to a ChemiDoc MP Imaging System (Bio-Rad).