Western blot analysis

The total proteins from mouse podocytes were extracted using radioimmunoprecipitation assay reagent (Beyotime Institute of Biotechnology, Haimen, China) supplemented with a protease inhibitor cocktail (Roche Molecular Diagnostics, Pleasanton, CA, USA). Subsequently, 40 µg extracted protein was separated by 8–15% SDS-PAGE, and transferred to 0.22-µm polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% milk in TBS with Tween-20 at room temperature for 2 h and subsequently incubated overnight at 4°C with microtubule-associated proteins 1A/1B light chain 3B (LC3)I, LC3II (cat. no. 4108; 1:1,000), autophagy protein 5 (Atg5; cat. no. 12994; 1:1,000), ubiquitin-like modifier-activating enzyme ATG7 (Atg7; cat. no. 8558; 1:1,000), caspase 3 (cat. no. 9662; 1:1,000), cellular tumor antigen p53 (p53; cat. no. 2524; 1:1,000), apoptosis regulator BAX (Bax; cat. no. 2772; 1:1,000), apoptosis regulator Bcl-2 (Bcl2; cat. no. 3498; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000) antibodies (all from Cell Signaling Technology, Inc., Danvers, MA, USA). Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. 7077 and 7076; 1:5,000; Cell Signaling Technology, Inc.) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a Western Blotting Detection and Imaging system (Bio-Rad Laboratories, Inc.). Enhanced chemiluminescence chromogenic substrate was quantified by densitometry (Quantity One 4.6 software; Bio-Rad Laboratories, Inc.).