Acrylamide gradient gel

To identify and estimate the levels of respiratory supercomplexes, homogenates from cortices were treated with digitonin (ratio 1:8, protein/digitonin; Roche), and mitochondrial complexes were separated by BN–PAGE in 3%–12% acrylamide gradient gels (Invitrogen) (67 (link), 68 (link)). To determine the levels of isolated respiratory complexes, cortex homogenates were treated with Dodecylmaltoside 1% final concentration (MilliporeSigma) and separated by BN-PAGE in 4%–16% acrylamide gradient gels (Invitrogen). 10 μg of protein was separated by PAGE, transferred to a PVDF membrane (Bio-Rad), and incubated sequentially with antibodies against several subunits of the different mitochondrial respiratory complexes. To detect the activity of CI in gel, mitochondrial complexes treated with Lauryl Maltoside (MilliporeSigma) and separated in 4%–16% gels were incubated with 14 mM NADH and 1 mg/mL nitroblue tetrazolium in Tris-HCL 0.1 M, pH 7.4, and incubated at 37°C for approximately 1 hour.

Eight micrograms of mitochondrial protein were supplemented with Laemmly loading buffer and separated on 10-comb precast 10% [w/v] to 20% [w/v] acrylamide gradient gels (Life Technologies, Darmstadt, Germany). Proteins were transferred to reinforced nitrocellulose membranes (Optitran BA-S 83, GE Healthcare, Freiburg, Germany) using semi-dry blotting (Trans-Blot SD Cell, Biorad, Munich, Germany) for 90 min in transfer buffer [37 mM glycine, 130 mM tris(hydroxymethyl)aminomethane, 0.275% [w/v] SDS, 20% [v/v] methanol]. For cytochrome c quantitation monoclonal antibodies directed against pigeon cytochrome c (7H8.2C12, Pharmingen, San Diego, CA, USA) in a 1:1000 dilution were used to probe the western blots. Signal generation was then achieved using anti-mouse secondary antibodies conjugated with horseradish peroxidase (HRP) via chemiluminescence in a buffer containing 100 mM Tris pH 8.5, 0.23 mM p-coumaric acid, 1.25 mM luminol, 0.00015% [v/v] H₂O₂. Quantitation of signals from three biological replicates was performed using the ‘image J’ software. For SDH1-1 quantitation, polyclonal antibodies (Peters et al., 2012 (link)) in a 1:1000 dilution were used and subsequently detected via biotin-conjugated goat-anti-rabbit secondary antibodies. Chemiluminescence signals of three biological replicates were generated by incubation with avidin-conjugated HRP via as described above.

Cells were harvested on ice by washing with cold PBS followed by lysing in a buffer containing 100 mM NaCl, 10 mM Tris-Cl, pH 7.6, 1 % (v/v) Triton X-100 and Complete protease inhibitor cocktail (Roche). Triton-insoluble material was sedimented by centrifugation at 20,000 g for 10 min at 4 °C. Supernatants were mixed with 5X SDS-PAGE sample buffer supplemented with DTT, then heated at 55 °C for 10 min prior to running in 4–20 % acrylamide gradient gels (Life Technologies and Bio-Rad). After SDS-PAGE, proteins were transferred onto PVDF membranes (EMD Millipore), blocked in 5 % non-fat milk (dissolved in PBS containing 0.1 % Tween-20), and probed with HA and CHC antibodies at 1:2,500 and 1:10,000, respectively. Blots were developed using enhanced chemiluminescence and imaged on a ChemiDoc digital imager (Bio-Rad). Protein signals were quantified using ImageJ (NIH). For overall TREM2 expression analysis, the TREM2 signals derived from cell lysates were first normalized to the corresponding CHC signal, then calculated as a fraction of the WT signal.