Fig. 6: Investigating regulatory functions of β-pS182/184.

A COS7 cells, transfected to express AMPK α1β1γ1 or α1β2γ1 (WT and β-S182/184 mutants), were grown in complete growth media. Prepared lysates were immunoblotted as indicated. B Sensitivities of AMPK α1β1γ1 (WT or β1-S182A mutant), FLAG-purified from COS7 cells incubated in complete growth media, to AMP (100 μM) were determined by radiolabelling of SAMS peptide substrate. Data presented as mean AMPK activity (nmol.min⁻¹ mg⁻¹) ± SEM, n = 4. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Kinetics of FLAG-purified AMPK α1β1γ1 (WT or β1-S182A mutant) from COS7 cells in response to increasing doses of (C) AMP or (D) A-769662 were determined by radiolabelling of SAMS peptide substrate. Data presented as average fold change in AMPK activity (nmol min⁻¹ mg⁻¹) vs. basal ± SEM, n = 4. E Lysates were prepared from cycloheximide-treated (CHX; 1 μM for up to 12 h) HEK293T cells, expressing AMPK α1β1γ1 (WT or β1-S182A), and immunoblotted as indicated. Data presented as average fold change in β1/actin protein content vs. untreated ± SEM, n = 3. Statistical analyses were performed for WT vs. β1-S182A at each time point by unpaired t test. Representative immunoblots from three independent experiments are shown.