Protease inhibitor and phosphatase inhibitor 2 3 cocktails

Subcellular fractionation was performed as described previously³⁶ . Briefly, RCN were harvested and washed in ice-cold phosphate-buffered saline. The cell suspension was centrifuged at 500 × g for 15 min at 4 °C. The cell pellet was resuspended for 10 min on ice in digitonin lysis buffer (20 mM HEPES, pH 7.4, 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 250 mM sucrose, 200 μg/mL digitonin, and protease inhibitor and phosphatase inhibitor (2,3) cocktails (Sigma-Aldrich, St. Louis, MO). Cells were passaged 20 times through a 22G needle. The lysate was centrifuged at 1000 × g for 5 min at 4 °C to pellet the nuclei. The supernatant was transferred to a new tube and centrifuged again at 12,000 × g for 10 min at 4 °C to pellet the mitochondria. The resulting supernatant, representing the cytosolic fraction, was recovered. Nuclear and mitochondrial lysates were prepared in RIPA buffer (Cat #R3792, Teknova, Hollister, CA) with protease inhibitor and phosphatase inhibitor (2,3) cocktails (Sigma-Aldrich). All steps were performed on ice. Pooled nuclear, cysotolic and total lysates were probed via electrophoresis and western blot for COX IV to identify mitochondrial content and Lamin to identify nuclear content to verify fractionation procedure.