Lysates were either made from mitochondrial fractions and whole cells. Fractions were collected in sucrose buffer (50 mM sucrose, 10 mMKCl, 20 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA [Sigma, E8145], protease inhibitor [Roche, 4693132001], pH 7.4) on ice. To break the plasma membrane, the cell suspension was passed through a 26-gauge needle (Sigma, Z192392) ~30 times. Separation of the mitochondrial and cytoplasmic fractions was performed using differential centrifugation, as shown previously [60 (link)]. Primary antibodies: ACTB (Abcam, ab8826) 1:5000, ATP5B (Abcam, ab14730) 1:5000, ERK1/2 (Cell signalling, 9102S) 1:1000, pERK1/2 (Y202/Y204) (Cell signalling, 9101) 1:500, SDHA (Abcam, ab109865), TSPO (Abcam, ab109497) 1:4000, USP30 (Enzo, MBL-PW0975) 1:1000, VDAC1 (Abcam, ab14734) 1:2000, 1:10000 Vinculin (Abcam, ab129002) and 1:1000 TFEB (Cambridge Bioscience, A303-673A). Imaging was carried using an ECL based method (GE, RPN2133) on a a Bio-Rad ChemiDoc MP Imaging System, following incubation for 1 h with either anti-rabbit conjugated HRP 1:4000 (Dako, P0447), or rabbit anti-mouse conjugated HRP (Dako, P0448), or goat anti-rat IgG conjugated HRP 1:4000 (Abcam, ab97057). Densitometric analysis was performed using the ImageJ software. Values were normalised to ACTB loading control for whole-cell lysates and ATP5B for mitochondrial fractions.
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