Pierce 660 protein assay

Dorsal striata from fresh brains were dissected and stored at −80°C. Synaptosomal fractions from bilateral regions were prepared for individual animals (for tgDISC1 and WT; n = 10 each), using a microscale discontinuous sucrose gradient modified from previous protocols (Hahn et al., 2009 (link); Sialana et al., 2016 (link)). Collected synaptosomes from 1.25/1.0 M sucrose interface were diluted with 10 mM HEPES, divided into two and pelleted at 15,000× g for 30 min. Pelleted synaptosomal samples were reconstituted in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 50 mM TEAB supplemented with protease inhibitors) for LCMS analyses and SDS buffer (1.5% SDS, 100 mM NaCl, 20 mM Tris supplemented with protease inhibitors) for WB analyses and were sonicated for 1 h. Protein amounts were estimated using the Pierce 660 protein assay or BCA protein assay (ThermoFisher Scientific).

Cell lysates were prepared using RIPA buffer supplemented with sodium orthovanadate, sodium fluoride and phenyl methyl sulphonyl fluoride (1 mM each). Total protein was quantified using the Pierce 660 Protein Assay (Thermo Scientific, Waltham, MA, USA) and equal amounts of protein loaded on to SDS-PAGE gels. Western blotting was carried out using standard protocols. Blots were probed with primary antibodies for HIF1α (ab279654), SOX9 (ab185966), GLUT1 (ab128033), LDHA (ab47010), IDH2 (ab129180) and PGC1α (ab191838) (Abcam, Cambridge, UK).

Samples were processed by addition of lysis buffer (2.1% SDS, 5% BME, 65 mM Tris–HCl, 0.1% Triton X-100, pH 6.8) containing protease and phosphatase inhibitors on ice. Samples were homogenized using a NextAdvance™ bullet blender for 2 cycles, and then sonicated. Samples were then centrifuged at 14,000×g for 12 min to remove debris. Supernatant was collected and total protein content was measured using the Pierce™ 660 Protein Assay from ThermoFisher Scientific. Samples were prepared for western blot by addition of Laemmli buffer and denatured by 90 °C heat for 5 min before SDS-PAGE and immunoblot. Proteins were resolved using 10–20 ug of protein loaded in 4–16% acrylamide stain-free gels from Biorad. After SDS-PAGE, the gels were imaged for protein in the stain-free gel before transfer. Proteins were then transferred to LF-PVDF membranes and blocked using T20 protein blocking buffer for 1 h. Primary antibodies were diluted at a 1:500 concentration in 5% milk solution and applied overnight at 4 °C. Following primary incubation, membranes were washed 3 times in PBST and secondary antibodies were applied at a 1:4000 concentration in 5% BSA-TBST for 2 h. Membranes were then washed 4 times and imaged. All western blot data was normalized to actin as a loading control. Each western blot sample was a biological replicate from an individual animal.