Sc 50132

Immunoblotting was performed with anti-CPT1A (1/1,000) (ab128568; Abcam), anti-CPT1B (1/1,000) (ab134988; Abcam), anti-CPT1C (1/1,000) (ab87498; Abcam), anti-CTP2 (1/1,000) (ab181114; Abcam), anti-heat-shock protein-60 (HSP60) (1/1,000) (ab46798; Abcam), anti-TOMM20 (1/1,000) (ab56783; Abcam), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-UQCRC2 (1/1,000) (ab14745; Abcam), anti-GFAP (1/500) (G6171; Sigma), anti-NDUFB8 (1/1,000) (ab110242; Abcam), anti-NDUFA9 (1/1,000) (ab14713; Abcam), anti-SDHA (1/1,000) (ab14715; Abcam), anti-MTCO1 (1/1,000) (ab14705; Abcam), anti-COX IV (1/1,000) (ab16056; Abcam), anti-Iba1 (1/1,000) (019-19741; Wako), anti-MAP2 (1/1,000) (ab32454; Abcam), anti-OLIG2 (1/1,000) (ab109186; Abcam), anti-PDHA1 (1/1,000) (no. 3205; Cell Signaling), anti-phosphoSer²⁹³-PDHA1 (1/1,000) (no. 31866; Cell Signalling), anti-β-Tubulin III (1/300) (T2200; Sigma) and anti-β-actin (1/30,000) (A5441; Sigma).

Immunoblotting was performed with anti- C-subunit of ATP synthase (SCMAs) (1/1000) (ab181243; Abcam), anti-VDAC (1/666) (PC548; Calbiochem), anti-heat-shock protein-60 (HSP60) (1/666) (ab46798; Abcam), anti-PINK1 (1/500) (sc-33796; Santa Cruz Biotechnology), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-CDK5 (1/500) (sc-6247; Santa Cruz Biotechnology), anti-PFKFB3 (1/500) (H00005209-M08; Novus Biologicals), anti-p25/35 (1/666) (2680; Cell Signalling), anti-caspase-3 (1/2000) (9661S; Cell Signalling), anti-CLN7 (1/500) (donated by Dr. Stephan Storch), anti-Parkin (1/100) (sc-32282; Santa Cruz Biotechnology), anti-LC3B (1/1000) (2775; Cell Signaling), anti-GFAP (1/500) (G6171; Sigma), anti-β-Tubulin III (1/500) (ab18207; Abcam) and anti-β-Actin (1/30,000) (A5441; Sigma).

The extracted and solubilized mitochondria were separated by electrophoresis in native gels (NativePAGE Novex 3–12% Bis-Tris Protein Gels, Thermo Scientific) in order to evaluate the arrangement of the complexes in the electron transport chain. The mitochondrial samples were loaded in 10% (v/v) loading buffer (5% (v/v) Serva-Blue G; 1 M 6-aminohexanoic acid). The electrophoresis was carried out at 100 V for 1 h in the presence of blue cathode buffer (50 mM tricine; 15 mM Bis-Tris/HCl; 0.02% (w/v) Coomassie Brilliant Blue G, pH 7) in the cathode chamber, and anode buffer (50 mM Bis-Tris, pH 7) in the anode chamber, followed by buffer replacement (removal of Coomassie blue dye) in the cathode chamber and electrophoresis at 40 V overnight at 4 °C. The gel containing the separated proteins was then incubated at 4 °C in carbonated transfer buffer (10 mM NaHCO₃; 3 mM Na₂CO₃·10 H₂O, pH 9.5–10) for 20 min with agitation to ensure that the gel was fully saturated with said transfer buffer. The transfer was then carried out onto polyvinylidene difluoride membranes (Thermo Scientific) (previously activated with methanol) at 60 V for 90 min at 4 °C in the Mini-Transblot system (Bio-Rad, USA). The membranes were then subjected to normal western blot protocol by using anti-NDUFS1 primary antibody (1:500) (Sc-50132, Santa Cruz) and anti-goat (1:10000) (ab6741, Abcam) as a secondary antibody.