Ab90085

Cultured H9c2 cardiomyocytes were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton. Cells were blocked with 3% BSA and incubated with 1000-fold diluted primary antibody against SMAD7 (ab90085; Abcam; Cambridge, MA, USA) overnight, and then stained with fluorochrome- conjugated secondary antibody for another 60 min. Cells were mounted in Vectashield mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI) to visualize nuclei. Images were captured using a fluorescence laser scanning confocal microscope (FV1000, Olympus, Tokyo, Japan).

Cells were harvested in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and centrifuged at 12,000×g for 15 min at 4°C. Whole cell lysate was used for SMAD7 detection. Cytosolic and nuclear fractions were prepared using standard nuclear and cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL, USA). Protein concentration was measured using the Bio-Rad method. Samples (20 µg protein) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 h prior to incubation with a primary antibody against SMAD7 (1∶1000; ab90085; Abcam), NF-κB p65 (1∶1000; ab7970; Abcam), GAPDH (1∶2500; ab7970; Abcam) or lamin B1 (1∶1000; #13435; Cell Signaling Technology; Boston, MA, USA) at 4°C overnight, followed by incubation with an appropriate peroxidase-conjugated secondary antibody (1∶1000 dilution). Signal was visualized by chemiluminescence (Odyssey Li-COR) using GAPDH as a control. In the case of nuclear NF-κB p65, lamin B1 was employed as the loading control. Band intensity was assessed using Quantity one 4.6.2 software.