Rabbit anti smad5

Protein samples were boiled in SDS/β-mercaptoethanol sample buffer, and 20 μg of protein from each sample was loaded on a gel. The antibodies used for western blotting were rabbit anti-CD9 (Abcam), rabbit anti-CD63 (Abcam) and rabbit anti-SMAD5 (Proteintech). Anti-GAPDH antibody (Proteintech) was used to detect GAPDH, which served as a loading control.

Protein expression in renal tissue and cultured cells was analyzed by Western blotting. Cells were lysed in ice-cold lysis buffer, and protein concentration was determined using the BCA Protein Assay Kit (Beyotime). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). Subsequently, the membranes were blocked with 5% non-fat milk for 1 h at room temperature and probed with primary antibodies, such as rabbit-anti-BMP-7 (1:1000), rabbit-anti-Smad1 (1:1000; Proteintech), rabbit-anti-Smad5 (1:1000; Proteintech), rabbit-anti-Phospho- Smad1/5^(Ser463/465) (1:1000, Cell Signaling Technology), rabbit-anti-E-cadherin (1:1000), rabbit-anti-Vimentin (1:1000; Santa Cruz), rabbit-anti-Collagen-III (1:1000), rabbit-anti-SnoN (1:1000), and rabbit-anti-β-actin (1:400; Proteintech), for 16 h at 4 °C. The signals were detected by chemiluminescence after incubation with appropriate secondary antibodies (). Images were acquired using the Bio-Rad gel imaging system (Bio-Rad, CA, USA), and the intensities of the immunoreactive bands were quantified using the Quantity One 4.6 software (Bio-Rad).