Mouse podocytes or rat glomeruli were collected and incubated in RIPA buffer (Beyotime) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice. Proteins were loaded on 12% SDS-PAGE gels and were transferred to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO, USA). Membranes were blocked in fat-free milk overnight at 4 °C and then incubated with primary antibodies overnight at 4 °C. Secondary antibody horseradish peroxidase-conjugated goat anti-rabbit/anti-mouse IgG (1:1,000; Beyotime Institute of Biotechnology, Haimen, China) was incubated 1 h at room temperature. The signals were visualized using enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA), and band densitometry was quantified using Quantity One version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Primary antibodies include PLK2 (1:1000), cleaved caspase-3 (1:500) from Abcam; Bcl-2 (1:400) and Bax (1:400) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); SIRT5 (1:1000), GAPDH (1:1500), and p53 (1:1000) from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Primary antibodies include PLK2 (1:1000), cleaved caspase-3 (1:500) from Abcam; Bcl-2 (1:400) and Bax (1:400) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); SIRT5 (1:1000), GAPDH (1:1500), and p53 (1:1000) from Cell Signaling Technology, Inc. (Danvers, MA, USA).