Ab14730

Sample proteins were quantified using a bicinchoninic acid (BCA) protein assay kit (Fisher Scientific, 13276818). Equal amounts of protein (30 µg for whole cell lysates/cytosolic fractions; 40 µg for mitochondrial fractions) were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Fisher Scientific, 10339574). The membranes were blocked in 3% non-fat dry milk in TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 h and incubated overnight with the primary antibodies at 4 °C: mouse α-p62/SQSTM1 (Abcam, ab56416) 1:20000; rabbit α-ubiquitin (Abcam, ab7780) 1:1000; mouse α-β-subunit (Abcam, ab14730) 1:15000; rabbit α-PARK2 (Cell signaling, 2132 S) 1:500; mouse α-actin (Abcam, ab8226) 1:5000; mouse α-MTCO-1 (Abcam, ab14705) 1:1000. Membranes were washed in TBS-T (3 × 5 mins at RT) and then incubated with corresponding peroxidase-conjugated secondary antibodies (Dako, P0447, P0448) for 1 h at room temperature. After a further washing step (TBS-T, 3 × 5 mins at RT), proteins were detected using an ECL Plus western blotting detection kit (Fisher Scientific, 12316992). ImageJ software was used to analyze immunoreactive bands by densitometry.

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.