Bcl 2 2876s

Antibodies used in the present studies included polyclonal antibodies to optic atrophy 1(OPA1) (80471S), LC3 (12741S), p62 (5114S), DRP1 (8570S), voltage‐dependent anion channel (VDAC) (4866S), cleaved caspase‐3 (9661S), Bax (2772S), Bcl‐2 (2876S), and PINK1 (6946S), all of which were purchased from Cell Signaling (Danvers, MA, USA). Mitofusin‐2 (MFN2) (Ab56889), Parkin (Ab15954), α‐synuclein (Ab1903), and phosphor S129 α‐synuclein (Ab51253) were purchased from Abcam (Cambridge, UK). TH (T1299), NeuN (ABN90), and β‐actin (A5441) antibodies were purchased from Sigma‐Aldrich. CCK8(C0038) was purchased from Beyotime Biotechnology (Shanghai, China). GFAP (SC‐71143) and Tom 20 (SC‐136211) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A320 (ALCAT1 inhibitor) was obtained from Perenna Pharmaceuticals Inc (San Antonio, TX, USA). ALCAT1 antibody was obtained from Dr. Arai (University of Tokyo, Japan).

Cells were harvested in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Protein lysate (20 μg) was resolved in SDS-polyacrylamide gels, transferred to PVDF membrane and then probed with antibodies specific to: folate receptor alpha (FR-α) (ab125030; Abcam, Cambridge, MA, USA); Cyclin D1 (72-13G), CDK4 (C-22), and PARP-1 (H-250) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); p21^WAF−1 (12D1) and Bcl-2 (2876S) (both from Cell Signaling Technologies, Danvers, MA, USA); and Caspase-3 (610322; BD Transduction Laboratories, San Diego, CA, USA). The membranes were next incubated with the respective secondary antibodies (Thermo Fisher Scientific). Anti-β-actin (AC-15; Abcam) and anti-β-tubulin (T5293; Sigma-Aldrich, St. Louis, MO, USA) antibodies were used as internal loading controls. The protein bands were quantitated using ImageJ software.