Anti mfn1

Mouse primary skeletal myotubes were solubilized in lysis buffer, as previously described^{6 (link),20 (link),27 (link),40 (link),63 (link),64 (link)}. For the co-immunoprecipitation assay^{20 (link),40 (link),63 (link)}, solubilized myotube lysate (80 or 100 µg of total protein) was used. The immunoprecipitate was subjected to immunoblot assays with anti-DHPR, anti-STIM1, or anti-GFP antibody (Abcam, Cambridge, MA, U.S.A., for detecting CFP-R429C). For the immunoblot assays, the solubilized myotube lysate (10 μg of total protein) was subjected to SDS-PAGE (8, 10, or 12% gel)^{6 (link),20 (link),27 (link),40 (link),63 (link),64 (link)}. Anti-RyR1, anti-SERCA1a, anti-CSQ1, anti-CaM1, anti-MG29, anti-MG53, anti-JP1, and anti-JP2 antibodies were obtained from Affinity BioReagents (Golden, CO, U.S.A.). Anti-TRPC1, anti-TRPC3, anti-TRPC4, and anti-TRPC6 antibodies were obtained from Alomone Laboratories (Jerusalem, Israel). Anti-Orai1, anti-STIM1, anti-STIM2, and anti-α-actin antibodies were obtained from Abcam. Anti-Drp-1, anti-Mfn1, anti-calcineurin and anti-CaMKII antibodies were obtained from Santa Cruz Biotechnology (Paso Robles, CA, U.S.A.).

Western blot was performed as previously described [17] (link). In brief, blocked membranes were incubated with anti-Beclin1, anti-LC3B (Cell Signaling Technology, Beverly, MA), anti-S100A7, anti-Mfn1, anti-Mfn2, anti-β-actin (Santa Cruz), or anti-DRP1 (BD Biosciences, San Jose, CA) at a dilution of 1∶1,000 or 1∶2,000. Immunoreactive bands were visualized using a chemiluminescent ECL detection kit (Pierce, Rockford, IL).

All antibodies utilized in this study: anti-CHCHD4 (Proteintech, #21090-1-AP); anti-β-Actin (Proteintech, #81115-1-RR); anti-α-SMA (CST, #19245S); anti-citrate synthetase (Abcam, # ab129095); anti-Vimentin (Abcam, #ab92547); anti-PCNA (Santa Cruz, #sc-56); anti-Ki67 (Abcam, #ab15580); anti-Opa1 (Santa Cruz, # sc-393296); anti-Drp1 (Santa Cruz, # sc-271583); anti-Mfn1 (Santa Cruz, #sc-166644); anti-SAM50 (Abcam, #ab246987); anti-DYKDDDDK Tag (CST, #14793); anti-HA (CST, #3724).

Adriamycin (ADR, catalog number D1515) and dihydroethidium (DHE, catalog number D7008) were obtained from Sigma-Aldrich (St. Louis, MO). Hyperoside (HPS) was purchased Zelang Biological Technology Company (Nanjing, China). Anti-nephrin (catalog number ab58968), anti-podocin (catalog number ab50339), anti-PGC-1α (catalog number ab54481), phosphor Anti-Drp1 (S637) (catalog number ab193216) and anti-VDAC (voltage-dependent anion channel, catalog number ab34726) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-Drp1 (catalog number sc-32898) and anti-Mfn-1 (catalog number sc-166644) antibodies were purchased from Santa Cruz (Santa Cruz, CA). anti-GAPDH was purchased from Sanying biotechnology (Wuhan, China, catalog number 10494-1-AP). All secondary antibodies for immunoblot analysis were from Zhongshan Golden Bridge Biotechnology (Beijing, China, catalog number ZB-2301). MitoSOX (catalog number M36008) and 2’,7’-dichlorofluorescein diacetate (DCFDA, catalog number C6827) were from Invitrogen (Carlsbad, CA, USA).

Cells were analyzed by western blotting using 4–12% SDS gradient gels (Invitrogen). The antibodies for the western blot analysis were as follows: anti-Drp1 (1:2000; Santa Cruz); anti-p-Drp1 (S637) (1:2000, Cell Signaling Technology, Beverly, MA, USA); anti-Mfn1 (1:1500; Santa Cruz); anti-OPA1 (1:500; Santa Cruz); anti-Fis1 (1:1000; Santa Cruz); anti-α2δ1 (1:1000, Santa Cruz); anti-β-actin (1:2000; Sigma-Aldrich); and anti-GAPDH (1:4000; Sigma-Aldrich). Immunoreactivity was analyzed by chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The chemiluminescence signal was observed with a digital image analyzer (LAS-3000; Fuji Film Inc., Tokyo, Japan).

The hippocampus or cells were lysed in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors and homogenized for 2 min. Protein concentrations were then measured using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, P0010). The loading volume was calculated according to a protein content per well of 40 μg; the experimental protocol was consistent with that of a previous study.²⁷ After blocking with 5% Albumin Bovine V (Solarbio, A8020) in Tris‐buffered saline containing 0.1% Tween 20 for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: anti‐Drp1, anti‐Fis1, anti‐OPA1, anti‐Mfn1, anti‐Mfn2 (Santa Cruz, CA, USA), anti‐p‐Drp1 (Ser637), anti‐p‐Drp1 (Ser616), anti‐cytochrome oxidase subunit IV (COXIV; Cell Signaling Technology), anti‐sequestosome 1, (p62; Cell Signaling Technology), anti‐microtubule‐associated protein 1 light chain 3β, (LC3B, Cell Signaling Technology and Abcam), and anti‐superoxide dismutase 2, (SOD2, Proteintech). Subsequent incubation was performed using goat anti‐rabbit/mouse secondary antibodies for 1 h at room temperature. The membranes were then imaged via enhanced chemiluminescence using an Odyssey infrared imaging system (LI‐COR Biosciences).

Whole kidneys were lysed in lysis buffer and centrifuged at 14000 g for 20 min at 4 °C. The supernatant was then collected. Nuclear and cytosolic renal proteins were extracted using a protein extraction kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Protein concentration was determined using a BCA protein assay kit. Total protein (50 μg) was separated during 12% SDS polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Burlington, USA) using a semi-dry transfer blotting apparatus. The membranes were incubated with primary antibodies for 18 h at 4 °C. The primary antibodies included rabbit anti-rat anti-CB1R (1:500) anti-p66shc (1:500), anti-Fis1 (1:500), anti-Mfn1 (1:500; Santa Cruz Biotechnology). After washing with TBST, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody goat anti-rabbit (1:5000 Santa Cruz Biotechnology, USA) for 60 min at 25 ± 1 °C. Add ECL chemiluminescence reagent, reaction 1 min at room temperature, X-ray film exposure development, fixing. Protein expression was measured with Image J (National Institutes of Health, Bethesda, MD, USA). Relative target protein expression was normalised to β-actin.