In aortic cells from the PKG I−/− mice the absence of PKG was confirmed by Western blot analysis, whereas in the control cells PKG I bands were easily detected.

Cells derived from the aortas of either mice having the PKG I gene disrupted or control mice were analyzed in the first passage to minimize any changes in PKG expression (expression of this gene is known to be affected by multiple passages of aortic smooth muscle cells). This suggests that the degree of PDE5 phosphorylation caused by 8-Br-cGMP was determined primarily by the level of PKG activation. SMCs were incubated with 1 mm 8-Br-cGMP for 60 min at 37 °C. After incubation PDE5 was quantitatively immunoprecipitated using mouse monoclonal PDE5 antibody.

To examine changes in PDE5 activity after phosphorylation a mouse monoclonal PDE5 antibody was used to precipitate total PDE5 activity (Fig. Lysates were incubated with phospho-PDE5 antibody overnight followed by incubation with protein A-agarose beads and then separated into supernatant and pellet. Cells were harvested in homogenization buffer A at different times after addition of PKA or PKG activators.

Phosphorylation of VASP, a well characterized substrate for both PKA and PKG, was also studied in these experiments as a control for the effectiveness of the analogs ( 15 ). VASP is a 46/50-kDa vasodilator-stimulated phosphoprotein that can be phosphorylated in response to either cAMP-elevating agents (prostaglandins or forskolin) or cGMP-elevating agents (nitric oxide donors). 8-Br-cGMP could stimulate phosphorylation of PDE5 by either PKG or the catalytic subunit of PKA but only at high concentrations. To measure cGMP PDE activity in the samples after phosphorylation, quick-spin columns were used to exchange the buffer and remove excess cGMP.

In vitro both PKG and PKA are able to activate/phosphorylate PDE5 in the presence of cGMP. As control experiments, PKA-induced phosphorylation of PDE5 in the presence of cGMP was completely blocked by PKI, whereas PKG-induced phosphorylation was not affected by the addition of PKI (data not shown). PDE1C and PDE5 immunoreactivity was determined in each fraction using Western analysis.

The PDE activity in each fraction was assayed with either 1 μmcAMP or 1 μm cGMP in the presence of either 1 mm EGTA or 1.0 mm CaCI2 and 4 μg/ml calmodulin. PDE5 is the major cGMP-hydrolyzing PDE expressed in human uterine SMCs. The presence of PDE1C in human uterine smooth muscle cells supports the observation that this enzyme is specifically expressed in proliferating human smooth muscle cells but not in non-human smooth muscle cells.

This antibody had a much higher sensitivity toward phosphorylated protein and could specifically recognize phosphorylated PDE5 but not dephosphorylated PDE5 analyzed at the ng range of loaded protein (Fig. 1 B). The specificity of the purified phospho-PDE5 antibody was tested against the same proteins using Western analysis. Membranes were stained with Ponceau S to visualize proteins (A andB) or treated with phospho-PDE5 antibody (C) for Western analysis.

Phosphorylated protein was separated by isoelectric focusing under native conditions (pH 5-8) (A) or SDS-PAGE (B) and transferred to nitrocellulose membrane.
18.07.2018 01:13:02

Maecenas aliquet accumsan

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos hymenaeos. Etiam dictum tincidunt diam. Aliquam id dolor. Suspendisse sagittis ultrices augue. Maecenas fermentum, sem in pharetra pellentesque, velit turpis volutpat ante, in pharetra metus odio a lectus. Maecenas aliquet
Or visit this link or this one