Figure 4: Effect of GRK2 inhibition on the Akt-NOS-NO signaling pathway. (a) The expression levels of Akt and eNOS mRNA were measured by real-time quantitative quantitative polymerase chain reaction (PCR). The data were presented as the fold change relative to the non-diabetic controls. (b) Akt, p-Akt (Thr308), p-Akt (Ser473), eNOS, p-eNOS (Thr495), and p-eNOS (Ser1177) protein expression were measured by western blot analysis. β-actin was used as a loading control. (c) Expression levels of eNOS and Akt protein were normalized to β-actin. (d) Densitometric ratios of phosphorylated Akt to total Akt and phosphorylated eNOS to total eNOS. (e) NOS activity was measured by fluorometric analysis. (f) Total NO level was measured by the colorimetric method. Results were reported as mean ± standard deviation (s.d.). *P < 0.05, the indicated group versus control group; #P < 0.05, the indicated group versus T2DM+vehicle group; n = 20 per group. All results are representative of three independent experiments. GRK2: G protein-coupled receptor kinase 2; T2DM: type 2 diabetes mellitus; Akt: protein kinase B; eNOS: endothelial nitric oxide synthase; p-eNOS: phospho-eNOS; p-Akt: phospho-Akt; NO: nitric oxide.