Mff 17090 1 ap

Primary antibodies used for Western Blotting were anti-GAPDH (MAB374; Millipore), VDAC1 (ab14734; Abcam), MFN1 (ab57602; Abcam), MFN2 (ab56889; Abcam), OPA1 (612607; BD Biosciences), FIS1 (10956-1-AP; Proteintech), MFF (17090-1-AP; Proteintech), DRP1 (Dlp1; 611112; BD Biosciences), involucrin (I9018; Sigma), profilaggrin (sc66192; Santa Cruz), HSP70 (ADI-SPA-810; Enzo life science), alpha-tubulin (sc8035; Santa Cruz). The following secondary antibodies were used: horseradish peroxidase-conjugated rabbit anti-mouse (P0161; DAKO) and horseradish peroxidase-conjugated goat anti-rabbit (PO448; DAKO). Hepes, D-Mannitol, Sucrose, Ethylenediaminetetraacetic acid (EDTA), Trizma Hydrochloride (Tris-HCL), Sodium chloride (NaCl), Igepal (NP-40), Sodium deoxycholate (DOC), Sodium dodecyl sulfate (SDS), Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma Aldrich.

The neuron cells were lysed in RIPA buffer (P0013K, Beyotime) containing both phosphatase inhibitor cocktail and protease inhibitor cocktail. Total proteins were electroporesed on 12% polyacrylamide gel containing sodium dodecyl sulfate and immediately transferred onto the PVDF membrane (ISEQ00010, Millipore). Blots were incubated overnight at 4 °C with the appropriate primary antibodies, followed by incubation with secondary antibody for 1 h at room temperature, and then visualized using ECL substrate solution (P0018, Beyotime). The following primary antibodies were used: Phospho-DRP1 (Ser616) (DRP1S616P) (3455, Cell Signal, 1:1000), FIS1 (10956-1-AP, Proteintech, 1:1000), MFF (1 7090-1-AP, Proteintech, 1:1000), and ACTIN (A2103, Sigma, 1:5000).

Samples were resolved by SDS-PAGE (10–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Bcl-x_L (54H6, 1:1,000 dilution, Cell Signaling; 2H12, 1:500 dilution, Invitrogen), COX IV (3E11, 1:20,000 dilution, Cell Signaling), Drp1 (D6C7, 1:1,000 dilution, Cell Signaling), Flag (66008-3-Ig, 1:1,000 dilution, Proteintech), GAPDH (sc-365062, 1:1,000 dilution, Santa Cruz biotechnology), GFP (sc-8334, 1:1,000 dilution, Santa Cruz biotechnology), HA (51064-2-AP, 1:1,000 dilution, Proteintech), Mff (17090-1-AP, 1:2,000 dilution, Proteintech), PARP (46D11; 1:1,000 dilution, Cell Signaling), RFP (3F5, 1:1,000 dilution, ChromoTek), SENP3 (D20A10, 1:10,000 dilution, Cell Signaling), and β-actin (A2228, 1:20,000 dilution, Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) or an HRP-conjugated VeriBlot secondary antibody (ab131366, Abcam; for immunoblotting involving IP samples) followed by enhanced chemiluminescence (GE Healthcare Amersham), or using fluorescent secondary antibodies (Thermo Fisher Scientific) by a LI-COR imaging system. Each immunoblot presented is representative of at least three independent experiments carried out using different cell populations.