Anti opa1 27733 1 ap

The selective CTSS inhibitor Z-FL-COCHO (ZFL) was purchased from Calbiochem Co. (Darmstadt, Germany). Temozolomide (TMZ, 76899) and BafilomycinA1 (BafA1) was purchased from Sigma-Aldrich (Missouri, USA). BAPTA-AM was purchased from Selleck Chemicals (Shanghai, China). Anti-Bax (#5023), anti-Bcl-2 (#3498), anti-cleaved caspase 3 (#9661), anti-PARP (#9532), anti-Atg3 (#3415), anti-Atg5 (#9980), anti-Atg7 (#2631), anti-Atg12 (#2010), and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3II (NB100-2220) and anti-Beclin 1 (NB110-87318) antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-CTSS (ab134157), anti-CD31 (ab28364), anti-MCU (ab121499), anti-MICU1 (ab190114), anti-MICU2 (ab101465), anti-EMRE (ab122209), and anti-p62/SQSTM1 (ab91526) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-Mff (17090-1-AP), anti-Drp1 (12957-1-AP), and anti-OPA1 (27733-1-AP) antibodies were purchased from Proteintech Inc(Rosemont, MA, USA).

Total protein was extracted from brain tissue or cultured cells with ice-cold RIPA buffer and protease inhibitor mixture (Solarbio). Equal amounts of protein per track were loaded onto 10% SDS-PAGE gels (Epizyme, Shanghai, China) for electrophoresis and then transferred onto PVDF membranes (Millipore) at low temperature. After blocking in 5% non-fat milk, the membranes were incubated overnight with primary antibody followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. The primary antibodies included anti-GAPDH (#10494-1-AP, Proteintech), anti-beta tubulin (#10094-1-AP, Proteintech), anti-TH (#AB152, Millipore), anti-CHIP (#2080, CST; Danvers, MA, USA), anti-Opa1 (27733-1-AP, Proteintech), anti-Drp1 (12957-1-AP, Proteintech), anti-Mfn1 (#13798-1-AP, Proteintech), anti-Mfn2 (#12186-1-AP, Proteintech), and anti-Fis1 (#10956-1-AP, Proteintech). Membranes were developed using the ECL luminescence reagent (#SQ101, Epizyme) and the protein bands visualized by enhanced chemiluminescence. Signal intensities were quantified with Image J software (NIH, Bethesda, MD, USA) and normalized to an internal control protein.