Protein Extraction and Analysis from HEK293T Cells

HEK293T cells were grown on 60 mm culture plates and transfected as described. For protein determination in whole-cell lysates, cells were washed with ice-cold PBS and lysed for 30 min on ice with RIPA buffer containing: sodium pyrophosphate 10 mM, Tritone X-100 2%, NaCl 100 mM, 5 mM EDTA pH 7.4, 5 mM EGTA pH 7.4, deoxycholate 0.5%, NaF 25 mM, sodium-orthovanadate 1 mM. Lysates were centrifuged at 4 °C for 20 min at 12,000 rpm and supernatant collected. For determination of protein expression in mitochondria, cell lysates were fractionated^{44 (link),106} . Protein concentration in the samples was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Equal amounts of protein (20ug) from lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot analysis (anti myc – Abcam ab18185 and anti VDAC1 [N-18] – Santa Cruz sc-8828) was performed as described previously^{23 (link),44 (link)}.