Ba1056

After 24 h of treatment with DDP or MMC, the drugs were removed and the RCC cells were cultured in medium for another 24 h. RCC cells that had been treated with drugs for 4, 12, and 48 h were collected, and T cells were collected after a 24-h incubation with RCC cells treated with DDP or MMC at a 10:1 ratio. Protein from these cells was extracted in lysis buffer (Beyotime Institute of Biotechnology). Equal amounts of protein (20 µg) after protein concentration determination by BCA on a microplate reader (ELx800™; BioTek USA) were loaded, separated on 10% sodium dodecyl sulfate (SDS) gels and transferred onto nitrocellulose membranes (Amersham; GE Healthcare). Then, the membranes were blocked with 5% nonfat milk at room temperature for 2 h and immunoblotted with anti-TGF-βR1 (3712), Smad2 antibody (5339; 1:1,000; Cell Signaling Technology, Inc.) or β-actin antibody (4967; 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. A horseradish peroxidase (HRP)-conjugated anti-rabbit or mouse antibody (BA1056; 1:10,000; Wuhan Boster Biological Technology, Ltd.) was reacted with the membrane at room temperature for 2 h. The immobilized antibodies were then detected by an ECL chemiluminescent reagent (EMD Millipore) and visualized with the ChemiDoc XRS system (Bio-Rad laboratories, Inc.).