Rabbit anti smad7

The protein concentrations in lysed and centrifuged remnant kidneys were measured using a bicinchoninic acid assay kit (Beyotime). The protein samples were mixed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and then boiled for 10 min. They were electrophoresed through SDS-PAGE gel, and then transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA). The membranes were incubated for 1 h in 5% skim milk on a shaker to block nonspecific protein binding. Primary antibodies against rabbit anti-TGF-β1 (1:1000; Abcam), rabbit anti-phospho-Smad 2/3 (p-Smad 2/3, 1:1000; Cell Signaling Technology), rabbit anti-Smad 7 (1:1000; Abcam), mouse anti-E-cadherin (1:1000; Cell Signaling Technology), rabbit anti-α-SMA (1:1000; Abcam), and mouse anti-β-actin (1:500; ZSGB-BIO) were incubated with the membranes at 4 °C overnight. The membranes were washed three times with Tris-buffered saline/Tween, and then incubated for 1 h with corresponding secondary antibodies (ZSGB-BIO). The ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Hercules, CA) was used for band detection, and positive immune reactions were analyzed using the Image J software.