Anti procaspase 3

Following the treatment of podocytes with 6.25, 12.5 or 25 µM BA, total protein was extracted from the conditionally immortalized mouse podocytes using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto a PVDF membrane (EMD Millipore) and blocked at room temperature with 5% fat-free powdered milk dissolved in tris-buffered saline (TBS) containing 0.1% Tween for 1.5 h. The membrane was incubated with the following primary antibodies at 4̊C overnight: Anti-SIRT1 (1:1,000; Cell Signaling Technology, Inc.), anti-cleaved caspase-3 (1:1,000; Santa Cruz Biotechnology, Inc.), anti-pro-caspase-3 (1:1,000; Santa Cruz Biotechnology, Inc.), anti-GAPDH (1:5,000; Cell Signaling Technology, Inc.), anti-p65 (1:1,000; Abcam) and anti-phosphorylated (p)-p65 (1:1,000; Abcam). Following the primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat no. ab7090; 1:2,000; Abcam) at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (EMD Millipore) and GAPDH served as a loading control for normalization.

Proteins were extracted from TCs using the pro‐prep protein extraction solution (iNtRON Biotechnology). The protein concentrations were determined using the DC protein assay kit (Bio‐Rad, USA). Approximately, 10 µL of each protein was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked with a blocking buffer containing 5% skim milk in Tris‐buffered saline. Cell cycle arrest was detected using anti-procaspase-3 (dilution, 1:1,000; Santa Cruz Biotechnology) and anti-cyclin D1 antibody (dilution, 1:1,500; Cell Signalling Technology, USA). An anti‐β‐actin antibody (dilution, 1:1,000; Santa Cruz Biotechnology) was used as a loading control. Immunoreactive bands were detected by chemiluminescence (Advansta, USA).

Western blotting was conducted on protein extractions that were obtained from the cell lysates. The protein concentrations were measured using a bicinchoninic acid (BCA) kit. Lysate proteins were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane. The membranes were blocked for 1 hour at room temperature using 5% nonfat milk in tris-buffered saline (TBS), then incubated at 4°C overnight with the following primary antibodies: anti-MYPT1 (1:1000; this and all subsequent antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Drp1 (1:1000), anti-Mfn1 (1:1000), anti-PGC-1α (1:1000), anti-NRF-1 (1:1000), anti-pro-caspase 3 (1:1000), anti-pro-caspase 9 (1:1000), and beta-actin (1:1000). After washing in TBS-Tween20, the membranes were incubated with secondary antibodies for 2 hours at room temperature. After washing, the membranes were incubated with enhanced chemiluminescence (ECL) reagents and scanned using a Bio-Rad Electrophoresis Image Analyzer (Bio-Rad, Hemel Hampstead, UK).^{19 (link),20}