Figure 5: Expression of the G87R and R139W mutants in HeLa cells decreases AMPK Thr172 phosphorylation in response to ionomycin. | Scientific Reports

Figure 5: Expression of the G87R and R139W mutants in HeLa cells decreases AMPK Thr172 phosphorylation in response to ionomycin.

From: Impact of Genetic Variation on Human CaMKK2 Regulation by Ca2+-Calmodulin and Multisite Phosphorylation

Figure 5

HeLa cells expressing either recombinant Flag-tagged CaMKK2 WT, G87R or R139W mutants were treated with vehicle (DMSO) or 5 μM ionomycin 5 min prior to lysis. Phosphorylation of Thr172 and total AMPK-α subunit protein was determined (shown on a cropped representative immunoblot) using rabbit pThr172 and mouse AMPK-α antibodies, and visualised simultaneously with goat anti-mouse IgG IRDye800 and goat anti-rabbit IgG IRDye680 fluorescently labeled secondary antibodies on a Infrared Imager. Expression of the Flag-tagged CaMKK2 constructs from the same lysates was determined on a separate immunoblot using rabbit anti-Flag and goat anti-rabbit IgG IRDye680 antibodies. The bar chart shows Thr172 phosphorylation quantified from the immunoblot, expressed as fold increase relative to the vehicle control. Data are presented as mean ± SEM; n = 3. Statistical analysis was performed by two-way ANOVA. *p < 0.01, vs the vehicle control within the same group; #p < 0.05 vs WT treated with ionomycin.

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