Pink1

The kidney tissues were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) to obtain proteins [22 (link)]. After the protein concentration was determined with the BCA kit (Beyotime Biotechnology, Shanghai, China), the proteins were separated by 10% of SDS-PAGE, followed by transferring to the polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked in blocking solution for 1 h at ambient temperature. The membranes were then incubated overnight at 4°C with primary antibodies provided by Cell Signaling, Boston, USA, including β-actin (#4970S, 1 : 1000), cleaved-caspase3 (#9661S, 1 : 1000), BAX (#5023S, 1 : 1000), Bcl-2 (#15071S, 1 : 1000), DRP1 (#8570S, 1 : 1000), MFN2 (#11925S, 1 : 1000), PINK1 (#6946S, 1 : 1000), Nrf2 (#12721S, 1 : 1000), Keap-1 (#8047S, 1 : 1000), and HO-1 (#86806S, 1 : 1000). The next day, after being rinsed for three times (10 min/time), the membranes were incubated with secondary antibodies (1 : 5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at ambient temperature, followed by a rinsing step for another three times (10 min/time). After dropping the developer on the membranes, the detection was performed using a chemiluminescence imaging system (Bio-rad).

Protein extraction was prepared from ESCs using RIPA buffer (Beyotime Biotechnology) with proteinase and phosphatase inhibitors. After being quantified by the BCA assay kit (Thermo Scientific), equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore). Membranes were blocked in 5% BSA (Sigma) at room temperature for 1 h, followed by incubation with primary antibodies against GAPDH (1:10000; Proteintech, 60004-1-Ig), GLS1 (1:1000; Abcam, ab93434), Collagen I (1:1000; Abcam, ab34710), α-SMA (1:1000; Abcam, ab7817), MFN1(1:1000; Abcam, ab57602), MFN2(1:1000; Abcam, ab56889), DRP1(1:1000; Abcam, ab56788), PINK1(1:1000; Cell Signaling Technology, 6946), p-p70-S6K (1:1000; Cell Signaling Technology, 9234), p-4E-BP1 (1:1000; Cell Signaling Technology, 2855) and p-S6 (1:1000; Cell Signaling Technology, 4858) overnight at 4 °C. The membrane was then incubated with HRP-conjugated goat anti-rabbit (1:10000; Proteintech, SA00001-2) or goat anti-mouse (1:10000, Proteintech, SA00001-1) secondary antibodies at room temperature for 1 h. Pageruler (ThermoFisher, 26,616) was used as the molecular marker. Protein expression was visualized with an ECL detection kit (Sigma-Aldrich). GAPDH was used as the loading control.

Total protein was extracted from cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with the anti-rabbit caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, PINK-1, Parkin, BNIP3, Beclin-1 and LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight after immersed into sealed liquid. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:1000, Cell Signaling Technology) labeled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:1000, Cell Signaling Technology) was used as a reference protein for normalization. The gray levels of the protein bands were examined by Image J software.