Tris acetate page gels

Cell extract proteins were resolved by Tris-Acetate PAGE gels (Invitrogen, Carlsbad, CA) and transferred onto a polyvinylidene difluoride (PVDF) membrane for western blotting. For Ub-VS labeling, we lysed cell pellets from control or treated cells with buffer (50 mM HEPES pH 7.4, 250 mM sucrose, 10 mM MgCl2, 2 mM ATP, 1 mM DTT and 1%NP-40) on ice for 30 min and removed debris by centrifugation. We labeled 25 μg of protein with 1 μM Ub-VS for 30 min at 37 °C. We resolved samples by SDS-PAGE and performed immunoblotting. For HA-Ub-VS labeling, we pretreated purified 19S (5 nM) with DMSO or 2.5, 5, 10, 15 or 25μM 1570 for 10 min at room temperature, which followed with labeling with 1 μM HA-Ub-VS for 30 min at 37 °C and immunoblotting.

For ubiquitin analysis, cell extract proteins were resolved by Tris-Acetate PAGE gels (Invitrogen, Carlsbad, CA) and transferred onto a polyvinylidene difluoride (PVDF) membrane for western blotting.

Cell extract proteins were resolved by Tris-Acetate PAGE gels (Invitrogen, Carlsbad, CA) and transferred onto polyvinylidene difluoride membranes, which were incubated overnight to antibodies, washed and incubated with HRP-conjugated anti-rabbit Ig (Amersham Biosciences, Little Chalfont, UK) for 1 h. Antibodies were used at the following dilutions: HIF-1α (1:200), β-actin (1:10,000), LC3 (1:1,000), BNIP3 (1:500), AMPK (1:1,000), phospho-AMPK (1:2,000), COX-1 (1:1,000), COX-IV (1:1,000), VDAC (1:1,000), p70 (1:1,000), phospho-p70 (1:1,000), eIF2-α (1:200), phospho-eIF2-α (1:200), caspase-4 (1:1,000), 4EBP1 (1:1,000) phospho-4EBP1 (1:1,000) and p62 (1:400). Peroxidase activity was developed by SuperSignal West Pico (Pierce Biotechnology). Unedited versions of Fig. 3d (LC3) and Fig. 4a (LC3 and BNIP3) are shown in Supplementary Fig. 8.