Anti drp1

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.).

Lung tissue sections were fixed in 4% paraformaldehyde, rinsed with PBS solution, incubated with 0.5% Triton X-100 for 15 min. at room temperature, and blocked with 10% serum. Primary antibodies included anti-SP-C; anti-Drp-1 and anti-Bax (Santa Cruz-Watsonville, CA, USA) were added at 4°C overnight. Tissue sections were subsequently rinsed with PBS and incubated with fluorescein labelled IgG antibody SP-C (Texas Red- labelled or FITC-labelled), Drp-1(Cy3-labelled) and Bax (FITC-labelled or Cy3-labelled). Hoechst 33258 (Sigma-Aldrich) was used for nuclear staining. After washing with PBS, tissue sections were mounted in glycerine and detected under a laser scanning confocal microscope from Leica Company.

Tissues were homogenized in lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, Tris-HCl 20 mM, pH 7.5) containing protease and phosphatase inhibitors. Proteins (15–30 μg/lane) were separated on 10–12% SDS-polyacrilamide gel and transferred to PVDF membranes (GE Healthcare). Blocking (5% non-fat milk, 30 min) and immunoreaction (o/n) were performed in TBST buffer (0.1% Tween, 150mM NaCl, 10mM Tris-HCl, pH 7.5) at room temperature. The primary antibodies were anti-Mitofusin1, anti-Mitofusin2, anti-DRP1 (all Santa Cruz), and anti-β-actin (BD Biosciences). Signals were visualized by horseradish peroxidase-linked secondary antibodies in enzyme-linked chemiluminescence (ECL, Millipore). Relative protein levels were normalized with β-actin levels on the same membrane.

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).

A western blot was performed as previously described.[22 (link)] Briefly, rabbit polyclonal anti-CnA antibody,[30 (link)] anti-p-Drp1 (phosphorylated form), and anti-Drp1 (unphosphorylated form) antibody[31 (link), 32 (link)] (1:200, Santa Cruz Biotechnology, Santa Cruz, USA) were used. Details of the procedures are further described in the S1 File.

Cells were lysed in RIPA lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM phenylmethanesulfonyl fluoride; 1 mM ethylenediaminetetraacetic acid (EDTA); 1% Triton X-100; 1% sodium deoxycholate; and 0.1% SDS) with the addition of Protease Inhibitor Cocktail (Roche Diagnostics, Penzberg, Germany) and Phosphatase Inhibitor Cocktail I (Sigma, St. Louis, MO, USA). The antibodies used included anti-COX2 (Abcam, Cambridge, UK), anti-β-actin (Cell Signaling, Danvers, MA), anti-OPA1 (Santa Cruz Biotechnology), anti-MFN2 (Santa Cruz Biotechnology), anti-DRP1 (Santa Cruz Biotechnology), anti-FIS1 (Santa Cruz Biotechnology), and peroxidase-labeled anti-rabbit IgG (H + L) secondary antibody (Abcam, Cambridge, UK). The signals were developed using ECL plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) using X-ray films.