Fig. 2.

GRK2 interacts with eNOS in mouse heart, an interaction that is increased after ischemia. A, B, Representative Western blots of GRK2 and eNOS co-immunoprecipitations at baseline (A), and post-ischemia-reperfusion (B). C, Quantification of the amount of cardiac eNOS immunoprecipitated by GRK2 in sham treated mice and mice after ischemia-reperfusion with eNOS/GRK2 (mean±SEM) values shown. *, P<0.05 (Mann-Whitney test, n=5 mice/group). D, Activation of eNOS induced by H₂O₂ treatment is blocked by co-expression of GRK2 in myocytes. eNOS was immunoprecipitated from NRVMs infected with an adenovirus containing eNOS (Ad-eNOS), with or without Ad-GRK2, and treated with H₂O₂. A representative Western blot for eNOS phosphorylated at Ser¹¹⁷⁷ (top panel) and total eNOS (bottom panel) is shown. E, Quantification of 5 separate experiments done as in (D). *, P<0.05, #, P<0.01 (Kruskal Wallis test). F, G, S-nitrosylation of GRK2 in GRK2 immunoprecipitates from hearts of GRK2 Tg and GRFK2/eNOS mice after ischemia-reperfusion, as determined by a Cys-NO antibody. A representative blot for SNO-GRK2 and total GRK2 after immunoprecipitation (F). The fold change (compared to sham-operated GRK2 Tg mice) of SNO-GRK2/GRK2 in GRK2 Tg and GRK2/eNOS mice after ischemia-reperfusion (G). *, P<0.05 (Mann-Whitney test, n=5 mice/group).