Figure 3.

CCCP treatment induces endogenous PINK1 kinase activity and Parkin E3 ligase activity in skeletal muscle cells. C2C12 myotubes were treated with DMSO (0.1%, 24 h) as a vehicle control (CTRL) or 10 μM of CCCP for up to 24 h. Total lysates were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBA^UBQLN1). Captured ubiquitylated proteins were subject to SDS‐PAGE and western blotting with antibodies specific to: phospho‐Ser65 ubiquitin (pSer65 Ub, to determine intracellular PINK1 kinase activity), CDGSH iron sulfur domain 1 (CISD1, to determine intracellular Parkin E3 ligase activity toward its substrate) and total ubiquitin (Ub, to verify ubiquitin enrichment). Representative images of n = 3 independent experiments are shown