FIGURE 4.

AMPKα siRNA abolishes the AICAR-induced increase in transport activity and cell surface abundance of Na⁺,K⁺-ATPase in L6 cells. L6 cells were transfected with siRNA against AMPKα or a scramble sequence as described under “Experimental Procedures.” Transfected cells were incubated in the presence or absence of 1 mm AICAR for 30 min. L6 cells were biotinylated with the cleavable biotinylation reagent sulfo-NHS-SS-biotin. An aliquot of the total cell extract was processed by a parallel SDS-PAGE. Biotinylated or total proteins were subjected to immunoblot analysis of: total AMPKα (A), pAMPK at Thr¹⁷² (B), pACC at Ser⁷⁹ (C), and cell surface α₁ Na⁺,K⁺-ATPase (D). Results are mean ± S.E. in arbitrary units, n = 7–10. *, p < 0.05 versus basal scrambled. E, ouabain-sensitive ⁸⁶Rb⁺ uptake assay was performed as described under “Experimental Procedures.” Results are mean ± S.E. n = 9–15. *, p < 0.05 versus basal. 2 mm ouabain was used to totally inhibit Na⁺,K⁺-ATPase.