Ecl kit

Adipose tissue samples were lysed with cold RIPA lysis buffer (0.5% Nonidet P-40, 0.1 M NaCl, 0.03 M HEPES with pH 7.6, protease inhibitors), and then the lysate was centrifuged at 13,000× g rpm, 4 °C for 15 min. The supernatants were collected and the concentration of the total proteins was measured using a BCA protein detection kit (Applygen Technologies, Beijing, China). Following this, 25 μg of protein were separated using SDS-PAGE gel and were moved onto NC membranes (Applygen Technologies, Beijing, China). Subsequently, the membranes were blocked with 2.5% nonfat milk and were incubated overnight with primary antibodies against anti-rabbit GPX4 (ABclonal, Wuhan, China), anti-rabbit SLC7A11 (ABclonal, Wuhan, China), anti-rabbit TFR1 (Invitrogen, Carlsbad, CA, USA), anti-rabbit L-ferritin (Abcam, Cambridge, UK), anti-rabbit H-ferritin (Abcam, Cambridge, UK), and anti-mouse β-actin (Cell Signaling Technology, Boston, MA, USA). After being washed three times, the membranes were treated with a corresponding horseradish peroxidase-conjugated secondary antibody (EASYBIO, Beijing, China). Finally, the protein bands were identified using an ECL kit (Applygen Technologies, Beijing, China). β-actin was served as the loading control.

The proteins of renal cortex tissue were lysed with Protein Extraction Kit (P0033, Beyotime, Jiangsu, China), the proteins of mitochondria and cytoplasmic were extracted with Tissue Mitochondria Isolation Kit (C3606, Beyotime, Jiangsu, China) and quantified by BCA protein assay kit (P1511, Applygen, Beijing, China). Total tissues lysates were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Afterward, the membranes were blocked with 5% BSA for 1 h at room temperature and followed by incubation with the primary antibodies anti-Parkin (SC-32282, Santa, Dallas, USA), anti-PINK1 (DF7742, Affinity, Ohio, USA), anti-LC3 (2775, CST, Boston, USA), SQSTM1/p62 antibody (5114, CST, Boston, USA), anti-Caspase-3 (9662S, CST, Boston, USA), anti-Cyt c (11940S, CST, Boston, USA), and anti-Nephrin (ab136894, Abcam, Cambridge, UK) overnight at 4 ℃. After washing, the corresponding HRPconjugated secondary antibodies were used. Finally, we used an ECL Kit (P1010, Applygen, Beijing, China) and a multi-functional imaging system to detect positive binding. Image-Pro Plus 6.0 software was used to quantify all protein bands.

Arteries or cells were placed into RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein samples (30 μg) were separated with 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). The membrane was blocked with blocking buffer (TBS, 0.1% Tween-20, 5% non-fat milk or 2% BSA) for 2 hours at room temperature and incubated with primary antibodies against eNOS (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-eNOS (ser¹¹⁷⁷) (1:1000, Abcam, Cambridge, MA, USA), arginase I (1:1000, Abcam, Cambridge, MA, USA), arginase II (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), UT-B (1:1000, a kindly gift from Dr. Trinh-Trang-Tan, INSERM, Paris, France), β-actin (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit IgG horseradish peroxidase (1:10000, Abcam, Cambridge, MA, USA) or goat anti-mouse IgG horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated for 60 minutes at room temperature the next day, respectively. The blots were developed with ECL kit (Applygen Technologies Inc, Beijing, China) and finally exposed to X-ray films. Relative protein expression levels were quantified by optical density analysis (Quantity-One software, Bio Rad Gel Doc 1000, Milan, Italy) and normalized to β-actin.

Cells were lysed in RIPA buffer on ice, and protein concentration was quantified by the BCA method (Sigma, MO, USA). 20 μg protein was resolved by SDS-PAGE and transferred onto PVDF membrane (Millipore, MA, USA). After brief blocking with 5% milk, the membrane was probed with primary antibodies: rabbit anti-Sox2 (#2748, Cell Signaling Technology, MA, USA), rabbit anti-Bmi1 (#6964, Cell Signaling Technology, MA, USA), rabbit anti-Lin28 (#3695, Cell Signaling Technology, MA, USA), rabbit anti-Nanog (#8822, Cell Signaling Technology, MA, USA), rabbit anti-ADGB (ab204085, Abcam, Cambridge, UK), rabbit anti-β-actin (#4970, Cell Signaling Technology, MA, USA) at 4 °C overnight. After washing, membranes were hybridized with secondary antibodies for another hour. The blots were detected with ECL Kit (APPLYGEN, Beijing, China) and visualized on LI-COR system (Biosciences, Lincoln, NE, USA).

Dispersion of lens capsules under a microscope was followed by extraction of the proteins with lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein extract was separated by electrophoretic 10% or 12.5% SDS‐PAGE (EPizyme, Shanghai, China). Then, it was transferred to PVDF membranes. The blots were incubated with the primary antibodies of LC3II/I (Abcam, ab192890), P62 (Abclonal, A19700), Lamp1 (Abclonal, A16894), Cln5 (Abclonal, A12886), Gla (Abclonal, A1700) and Lipa (Abclonal, A6385). The blots were incubated with a secondary antibody (ZSGB‐Bio, ZB‐2301), and then they were visualized using enzyme‐linked chemiluminescence using the ECL kit (Applygen, Beijing, China).