Functional assays for PRKAB2 in patient cells. (A). Left panels:Titrated whole cell extracts wereblotted for AMPKβ2 (encoded by PRKAB2) in wild-type (WT),Del and Dup containing LBCs. Right panels:Blots were re-probed with anti-β-tubulin. Graph: Densiometric quantification of AMPK-β2 expression from titrated extracts, going from low (black bar), intermediate (white) to higher (grey) amounts of protein, normalized to β-tubulin loading, from three separate determinations (a.u. arbitrary units). p = 0.01 for Del and p < 0.005 for Dup LBCs compared to WT. (B). AMPK subunit AMPK-β1, encoded by the PRKAB1 gene on chromosome 12q24.1, shows equivalent expression in the wild-type (WT), Del and Dup containing LBCs. β-tubulin was used to confirm equal loading. (C). AICAR-induced (2 mM) activation of the AMPK kinase was monitored using phosphorylation of the AMPKα subunit on threonine 172 (p-T172-AMPKα). Dup and Del containing LBCs exhibited higher levels of p-T172-AMPKα at the 0 time (untreated), relative to wild-type (WT). Only the 1q21.1 Del containing LBCs appeared to be unresponsive to AICAR-treatment here. Blots were re-probed with for native AMPKα to confirm loading. (D). AICAR-induced (2 mM) activation of AMPK was evaluated by monitoring phosphorylation of the AMPK substrate Acetyl-CoA Carboxylase on serine 79 (p-S79-ACC). Similar to p-T172-AMPKα, the Del containing LBCs appear unresponsive to the AICAR treatment. Blots re-probed for native ACC to confirm loading. (E). AICAR-induced activation of AMPK was also evaluated by phosphorylation of the AMPK substrate RAPTOR on serine 792 (p-S792-RAP) under identical conditions as in (B) and (C). Again, Del containing LBCs appeared somewhat unresponsive to AICAR. Blots re-probed for MCM2 to confirm loading.
Harvard et al. Orphanet Journal of Rare Diseases 2011 6:54 doi:10.1186/1750-1172-6-54