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. Author manuscript; available in PMC: 2009 Oct 13.
Published in final edited form as: Circ Res. 2009 Jan 8;104(4):496–505. doi: 10.1161/CIRCRESAHA.108.187567

Figure 2.

Figure 2

AMPK phosphorylates eNOS Ser635 in cultured ECs. Confluent BAECs were treated with various concentrations of AICAR for 15 minutes (A) or infected with Ad-AMPK-CA at different multiplicities of infection (MOI) for 24 hours (B). The control cells were infected with Ad-null virus at 50 multiplicities of infection. Cell lysates were analyzed by Western blotting with the indicated antibodies. C, BAECs were treated with AICAR at 1 mmol/L for 15 minutes or left untreated as controls. AMPKα was immunoprecipitated from cell lysates with the use of anti–pan-AMPKα at 1:20 or 1:15 dilution. Rabbit IgG was used as an IP control. The kinase activity of immunoprecipitated AMPKα was assayed with recombinant GST-eNOS (wild type [WT]), GST-eNOS (S1179A), or GST-eNOS (S635A) as substrates. Left, The expressed GST-eNOS is shown by Coomassie blue staining and Western blotting. The phosphorylation of GST-eNOS S1179 and S635 by the immunoprecipitated AMPKα was detected by Western blotting. The level of immunoprecipitated AMPKα and GST-eNOS used in the assays was also shown by immunoblotting. Data represent results from 3 independent experiments.