Figure 3. PIAS4 inhibits AMPKα1 activity in a SUMO E3 ligase-dependent manner.

(a) AMPKα1 immunoprecipitates from cells with the indicated AMPKα genotypes transfected with indicated siRNAs followed by treatment with vehicle (−) or AICAR (+) were used to phosphorylate recombinant GST–TSC2 (1,300–1,367) in vitro in the presence of [γ-³²P] ATP followed by SDS–PAGE and autoradiography (32P GST–TSC2) or WB analysis with antibodies as indicated on the left. (b) NIH3T3 cells transfected with vector control (−), Flag–PIAS4-WT (WT) or Flag–PIAS4-C335F (C335F) plasmids and 24 h later treated with vehicle (−) or 2 mM AICAR (+) for 2 h were lysed and analysed by SDS–PAGE and WB with antibodies indicated on the left. For PIAS4 blot, the asterisk denotes unspecific signals and the bracket denotes specific signals. (c) AMPKα1 immunoprecipitates from NIH3T3 cells transfected with non-targeting (−) or AMPKα1 (+) siRNAs and PIAS4 plasmids as indicated, and treated for 2 h with vehicle (−) or 2 mM AICAR (+) were used in kinase assays as in a with recombinant GST–TSC2 (1,300–1,367) control (WT) or S1345A mutant as substrates. (d) Lysates from immortalized MEFs transfected with the non-targeting (siNT) siRNA pool or two independent siRNAs against mouse Ubc9 (siUbc9-1 and siUbc9-2) and 72 h later treated with vehicle (−) or 2 mM AICAR (+) for 2 h were analysed by WB with indicated antibodies. Representative figures from two (b,d) or three (a,c) experiments are shown.