Figure 7.

AMPK activation by 991 induces TBK1 activation in a PINK1‐Parkin independent manner. A, 991 treatment induces TBK1 phosphorylation in C2C12 myotubes. C2C12 myotubes were serum starved for 4 h prior to the treatment of DMSO (0.1%) as a vehicle control (CTRL) or AMPK activator 991 at the indicated concentrations for 2 h. B, 991 activates AMPK signaling in C2C12 myotubes. Cells treated as in Figure 5A. C, 991‐induced TBK1 phosphorylation is diminished in AMPK ⍺1/⍺2 deficient cells. HEK293 wild type and AMPK ⍺1/⍺2 deficient cells were treated with DMSO (0.1%) as a vehicle control (CTRL) or AMPK activator 991 (10 μM) for 1 h and 2 h. Cells were serum starved for 2 h prior to treatment. D, AMPK activation does not induce PINK1‐Parkin signaling in skeletal muscle cells. C2C12 myotubes were serum starved for 4 h prior to the treatment of DMSO (0.1%) as a vehicle control (CTRL) or AMPK activator 991 at the indicated concentration for 2 and 24 h. As negative and positive controls, C2C12 myotubes were treated with DMSO (0.1%, 24 h, lane 7) or CCCP (10 μM, 24 h, lane 8), respectively, without serum starvation. For ubiquitin pulldown, total lysates were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBA^UBQLN1). Ubiquitin enriched extracts were analyzed by SDS‐PAGE and western blotting with the indicated antibodies. Representative images of n = 3 independent experiments are shown. Data are expressed as means ± SEM; *P < .05, **P < .01, ***P < .001, ****P < .0001 compared to CTRL. ^σ P < 0.05, ^δ P < 0.0001 compared to wild type 1 h. ^η P < 0.05, ^γ P < 0.0001 compared to wild type 2 h