Figure 5. Phosphorylation of p47^phox and p67^phox subunits.

(A) Cells were incubated with Hcy (100 μM) or without Hcy for various time periods. p47^phox and p67^phox proteins were immunoprecipitated (IP) using goat anti-p47^phox or p67^phox antibodies followed by Western-blot analysis (IB) using anti-phosphoserine antibodies to detect serine-phosphorylated proteins. The immunoblots were analysed by densitometry. Results were normalized to total p47^phox and p67^phox proteins in the immunoprecipitates and are means±S.E.M. for five separate experiments. The relative densitometric units for the ratio of phosphorylated to total p47^phox or p67^phox proteins in the control cells (incubated in the absence of Hcy) were expressed as 100%. (B) Cells were incubated for 30 min in the absence (Control) or presence of Hcy (100 μM). The membrane fraction was prepared for detection of p47^phox or p67^phox proteins by Western-blot analysis. β-Actin was used to confirm equal amount of protein loading for each sample. Representative blots were obtained from five separate experiments. *Significantly different from the control value obtained from cells incubated in the absence of Hcy (P<0.05).