Research Paper Volume 5, Issue 7 pp 515—530

Figure 3. Above-physiological ROS levels induce AMPK-mediated eNOS activation in Tet-OFF MHEC.

(A) Western blots (WB) analyses of MHEC protein lysates from two independent lines of NVF Tet-ON and Tet-OFF mice as indicated. WB was carried out using anti-phospho-AMPK (p-AMPK), anti-p-Akt (ser473), anti-p-eNOS (ser1179) and anti-T-Akt (total) antibodies. T-Akt was used as loading control. Right panels, bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant. (B) Protein extracts from Tet-OFF MHEC transfected with control siRNA (Scram-si) or si-AMPK were subject to Western blots as described in the Methods. Membranes were sequentially blotted, stripped and re-probed with anti-AMPK, anti-p-eNOS and GAPDH antibodies as shown. Representative blots of two independent experiments are shown. (C) NO production, as measured using citruline assay as described in Methods, was 2.1±0.32-fold higher in Tet-OFF MHEC compared to Tet-ON. Si-AMPK significantly inhibited NO production in Tet-OFF MHEC. *p<0.05.