Fig. 5.

Activated AMPK triggers β-TrCP1 degradation upon glucose deprivation. a Cells were cultured in high-glucose or glucose-free medium for the indicated time periods and then harvested for IB with anti-β-TrCP1, p-ACC, t-ACC, p-RSK, t-RSK, p-AMPK, t-AMPK, p-AKT, t-AKT, p-ERK1/2, t-ERK1/2, and Actin Abs. b Cells transferred to glucose-free medium were treated with or without 10 μM Compound C for 1 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-ACC, and Actin Abs. c Cells were pretreated with DMSO or 0.5 mM AICAR for 24 h, lysed under denaturing conditions and then subjected to pull-down by Ni-NTA beads. Pull-downs (top) and whole-cell extracts (bottom) were subject to IB with anti-HA and Actin Abs. SK-BR3 cells were transfected with siRNA targeting AMPKα or scrambled control siRNA for 48 h (d); AMPK WT or DKO MEFs were shifted to glucose-free medium for 2 h (e), treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-AMPK, t-AMPK, p-ACC, t-ACC, and Actin Abs. f SK-BR3 cells were transfected with siRNA targeting LKB1 or scrambled control siRNA. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, LKB1, p-AMPK, t-AMPK, and Actin Abs. g Human β-TrCP1 has a putative AMPK motif (75-SLRQTYNSCARL-86) that is highly evolutionarily conserved. Hyd: bulky hydrophobic residues (like L, I, M, F, and V); X^*: one of these sites is a basic residue. h H1299 cells were transfected with FLAG-β-TrCP1 WT or S82A mutant plasmid. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-FLAG and Actin. Densitometry quantification was performed with ImageJ, and the decay curves are shown (mean ± S.E.M., n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (b, d–f, h, right). GD: glucose deprivation; C.C: Compound C; LEX: longer exposure. “t-” denotes antibodies that recognize total protein rather than a specifically modified version