Figure 2.

CaMKKs mediate M3 mAChR-induced AMPK phosphorylation.A and B, cells were preincubated with STO-609 (1 μM) for 15 min and then stimulated with (A) PD 102807 (100 μM; t = 20 min) or (B) MCh (100 μM; t = 5 min). C and D, cells were transfected with siRNA against CaMKK1 or CaMKK2 and then stimulated with (C) PD 102807 or (D) MCh. AMPK and ACC phosphorylation was determined using phospho-specific antibodies. E, knockdown of CaMKK isoforms was confirmed by immunoblotting. Representative blots are shown. Loading was corrected for total AMPK or β-actin. Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗p < 0.0001 versus vehicle stimulation, #p < 0.05, ###p < 0.001 versus respective mock-transfected PD 102807 or MCh; one-way ANOVA followed by Bonferroni multiple comparison test. ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; CaMKKs, Ca²⁺/calmodulin-dependent kinase kinases; mAChR, muscarinic acetylcholine receptor; MCh, methacholine.