FIGURE 9.

PP2A^Ppp2r2d regulates AMP kinase activity in human vascular smooth muscle cells. A, HVSMCs were grown as described under “Experimental Procedures.” Cells were treated with control siRNA (Csi) or siRNA directed against PPP2CA or PPP2R2D, and protein level and phosphorylation status were determined by Western analysis. Actin was used as a loading control. B and C, cells were treated with control (Csi) or LKB1 siRNA (LKB1si), CAMKKβ siRNA (CAMKKsi), or LKB1 and CAMKKβ siRNAs. OA was added under the indicated conditions. Protein levels were determined using Western analysis. The level of Camkkβ activity was determined by assaying for the level of CamkII phosphorylation. The phosphorylation status of AMP kinase α was determined using antibodies directed against Thr-172. Actin was used as a loading control. D, 10% of the protein input used for co-immunoprecipitation assays. Actin was used as a loading control. E, extracts were incubated with the indicated antibodies (IP). Bound proteins were pulled down using Protein A-Sepharose and resolved by SDS-PAGE. Co-immunoprecipitated proteins were determined using Western analysis (WB).