FIGURE 7.

AICAR decreases the phosphorylation of Na⁺,K⁺-ATPase. Cells were pretreated with dimethyl sulfoxide (Basal), AICAR (1 mm for 30 min), or PMA (500 nm for 40 min). A, an autoradiogram of ³²P-labeled total proteins after SDS-PAGE and electrophoretic transfer is shown. B, Na⁺,K⁺-ATPase α₁-subunits were immunoprecipitated using Na⁺,K⁺-ATPase α₁-subunit antibody as described under “Experimental Procedures.” Immunoprecipitates (IP) were then processed by SDS-PAGE and subjected to immunoblot analysis using a Na⁺,K⁺-ATPase α₁-antibody. C, total proteins were subjected to immunoblot analysis with the McK1 antibody. The McK1 antibody recognizes the unphosphorylated α₁-subunits (on Ser¹⁸). Activation of PP2A was assessed by measurement of phosphorylation at Tyr³⁰⁷ (D) and methylation at Leu³⁰⁹ (E). Representative blots and densitometric analysis are shown. Results are mean ± S.E., n = 4–5. *, p < 0.05 versus basal. WB, Western blot.