Lds sample buffer

HeLa cells were cultured in 6-well plates for 24 h prior to incubation with either fresh culture medium, EBSS starvation medium, or fresh medium containing 10 μM oligomycin (Calbiochem), 4 μM antimycin A (Sigma) and 10 μΜ QVD (ApexBio) for the times indicated. For immunoblot analysis of cell lysates (Figs 1a, 5e; Supplementary Fig. 5n), cells were lysed in 1 × LDS sample buffer (Life Technologies) supplemented with 100 mM dithiothreitol (DTT; Sigma) and heated to 99 °C with shaking for 7–10 min. For isolation of crude mitochondrial fractions (Supplementary Fig. 3a, f, i), cells were homogenized in 20 mM HEPES (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. After centrifugation of the cell homogenates at 800× g for 10 min (4 °C), mitochondria in the post-nuclear supernatant were pelleted by centrifugation at 10,000×g for 20 min (4 °C). The mitochondrial pellets were lysed in 1× LDS sample buffer (Life Technologies) supplemented with 100 mM dithiothreitol (DTT; Sigma) and heated to 99 °C with shaking for 7–10 min. Approximately 25–50 μg of protein per sample was separated on 4–12% Bis-Tris gels (Life Technologies) according to manufacturer’s instructions, electro-transferred to polyvinyl difluoride membranes, then immunoblotted using antibodies as indicated. Uncropped data for immunoblots are provided (Supplementary Fig. 7).

Subcellular fractionation was performed as previously reported^{129 (link)}. Cell pellets were resuspended in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, and PIs: NaF, PMSF, 1× Halt Protease inhibitor cocktail), incubated 5 min on ice, and centrifuged for 5 min at 1300 × g at 4 °C. The supernatant was taken as the cytoplasmic extract and clarified by centrifugation. Nuclear pellets were washed in buffer A and resuspended in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and PIs). After 5 min on ice, chromatin pellets were centrifuged for 5 min at 1700 × g, 4 °C. The supernatant was collected as the soluble nucleoplasmic fraction. LDS sample buffer (Thermo Fisher Scientific) was added to 1× to cytoplasmic and nucleoplasmic fractions. Insoluble pellets were resuspended in 1× LDS sample buffer (Thermo Fisher Scientific) containing 5% β-mercaptoethanol and sonicated on a Bioruptor: high power, 30 s on, 30 s off, 5 min total (Diagenode). All samples were denatured for 5 min at 95 °C followed by cold shock.