Cw0100m

Western blotting was performed as previously described (21 (link)). Mouse kidney and cultured cells were lysed in RIPA lysis buffer. Anti-GAPDH (1:2000 dilution, CW0100M, CWBio, China), anti-cleaved caspase 3 (1:1000 dilution, ab32503, Abcam, Cambridge, UK), anti-Bcl2 (1:1000 dilution, cst2870, Cell Signaling Technology, USA), anti-SIRT1 (1:1000 dilution, GT1189, Genetex, China),anti-KIM-1(0.25ug/ml, AF1817, RD systems, Canada), anti-PGC-1a(1:1000, # 2178, cell signaling technology, USA) and anti-Bax (1:1000 dilution, ab193349, Abcam Cambridge, UK) antibodies were used in this study. Experiments were repeated three times.

The relevant protocols were reported previously^{10 (link)}. Briefly, MSCs were cultured with HD serum or AS serum for 2 days and lysed with RIPA buffer containing protease and phosphatase inhibitors for 30 min on ice. The lysates were collected and centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentrations of the lysates were detected with the BCA Protein Assay Kit (CWBIO, CW0014S), and equal amounts of proteins were mixed with a 5× sodium dodecyl sulfate (SDS) loading buffer. Then, the proteins were separated via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). After being blocked with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies against p53 (1:1000, Cell Signaling Technology, 2524S), p21 (1:1000, Cell Signaling Technology, 2947S), p16 (1:1000, Cell Signaling Technology, 80772S) or GAPDH (1:2000, CWBIO, CW0100M) at 4 °C overnight. Then, the membranes were incubated with HRP-conjugated secondary antibodies (1:3000, Boster, BA1050 and BA1054) for 1 h and detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore WBKLS0500).

The experiment was performed as previously described.¹⁴ First, RIPA buffer was prepared, and cells were lysed for 30 min on ice. Then, the cell lysates were collected and centrifuged at 14,000 rpm for 30 min at 4°C. After that, the protein concentrations were measured, and equal amounts of proteins were mixed with loading buffer. The proteins were separated via electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). Then the membranes were blocked with 5% non‐fat milk solution for 1 h. Thereafter, the membranes were incubated with primary antibodies followed by secondary antibodies. Finally, protein levels were detected using Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and analyzed with ImageJ.
The following antibodies were used for western blotting: anti‐Osterix (Abcam, ab209484, 1:800); anti‐OCN (Abcam, ab93876, 1:800); anti‐AGO2 (Abcam, ab186733, 1:1500); anti‐RUNX2 (Cell Signaling Technology, 12556S, 1:1000); anti‐Collagen I (ColI; Abcam, ab260043, 1:1500); anti‐IRF2 (Abcam, ab124744, 1:2000); anti‐YY1 (Abcam, ab109228, 1:2000); anti‐GAPDH (CWBIO, CW0100M, 1:3000); and HRP‐conjugated secondary antibodies (Boster, 1:5000).