Induction of HO-1 protein and AMPK activation by OA-NO₂.

Abstract

<p><b>A</b>) BAECs were incubated with OA-NO₂ at the indicated concentrations or with BSA (vehicle) for 16 h, and western blot analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a> to detect HO-1 protein expression and AMPK phosphorylation at Thr172. The blot is representative of those obtained from three separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *<i>p</i><0.05 <i>vs.</i> control. <b>B</b>) BAECs were incubated with 2.5 µM OA-NO₂ for the indicated times, and western blotting was performed as above. The blot is representative of three blots obtained from three separate experiments. *<i>p</i><0.05 <i>vs.</i> corresponding control. <b>C</b>) Confluent BAECs were exposed to vehicle or OA-NO2 (2.5 µM) for 16 h. AMPKα was immunoprecipitated from cell lysates (1 mg) with a specific antibody. AMPK activity was assayed by ³²P-ATP incorporation into the SAMS peptide. *<i>p</i><0.05 <i>vs.</i> control. <b>D</b>) BAECs were incubated with the indicated concentrations of OA for 16 h. Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. <b>E</b>) BAECs were infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). Infected and non-infected cells were treated with 2.5 µM OA-NO₂ for 16 h. AICAR and metformin were used as positive controls. The blot is representative of three blots obtained from three separate experiments.</p