Anti drp1

Hoechst 33342 (Invitrogen) was used at a concentration of 100 ng/ml. MG-132 (Cayman Chemical) was used at 10 μM and 3 Methyladenine (Sigma-Aldrich) at 5 nM. Primary antibodies used for immunofluorescence experiments were the following: anti-MAVS (Cell Signaling, 1:500), anti-Flag (Sigma, 1:500), anti-Tomm20 (Santa Cruz, 1:1,000), and anti-DRP1 (BD Biosciences, 1:200). The secondary antibodies used for immunofluorescence were the following: polyclonal anti-mouse Ig Alexa488 or Alexa568 conjugated (Invitrogen, 1:1,000), polyclonal anti-rabbit Ig Alexa488, Alexa568, or Alexa647 conjugated (Invitrogen, 1:1,000), polyclonal goat anti-rabbit Ig ATTO647N (Active Motif, 1:1,000), and polyclonal goat anti-mouse IgG MegaRed 520 (Sigma, 1:100). Primary antibodies used for immunoblotting were the following: rabbit anti-MAVS, ATG5 and DRP1, mouse anti-GFP, and IFN-α (Cell Signaling), mouse anti-PCPB2 and anti-AIP4 (Santa Cruz Biotechnology), monoclonal anti-Myc (Clontech Laboratories), mouse anti-Flag and rabbit anti-LC3 (Sigma-Aldrich), rabbit anti-K48-linked polyubiquitin chains (Millipore), mouse monoclonal anti-IFN-β (Biolegend), and mouse anti-actin conjugated to horseradish peroxidase (HRP, Sigma-Aldrich). For immunoprecipitations monoclonal mouse anti-Myc, anti-PCPB2 or anti-GFP and Protein G PLUS–Agarose (Santa Cruz Biotechnology) were used.

Osteoblasts cultured in different conditions were lysed with RIPA buffer. Proteins were electrophoresis by SDS-PAGE and transferred to membrane. Anti-phospho-Drp1 (1:3000, cell signaling), anti-Drp1 (1:3000, BD Science) and anti-β-actin (1:5000, Sigma) were used as primary antibodies. The binding sites of primary antibody were visualized with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Invitrogen) or anti-mouse IgG antibody(1:5000, Invitrogen), followed by the addition of ECL substrate. The immunoreactive band relative to optical density was determined by using NIH Image J software (public domain) and normalized with β-actin levels.