Fig. 7. Effect of H₂O₂ on c-Abl phosphorylation.

(A) Immunoprecipitation of GFP-tagged Abl was performed using anti-GFP antibodies and 250 μg of proteins obtained from lysates of K562/NOX5 cell expressing either kinase-active GFP-c-Abl or the kinase-dead GFP-c-Abl. The immunoprecipitated proteins were immunoblotted with phosphotyrosine or c-Abl antibody. K562/NOX5 cells were used as a negative control. (B) Immunoprecipitation experiment were performed as in A, except that the K562/NOX5 cells expressing wild-type GFP-c-Abl were either untreated or incubated with BAPTA (50 μM) and then treated (or not treated) with 100 μM H₂O₂ for 10 minutes. In the GFP immunoprecipitates, the levels of phosphorylated Abl proteins (GFP-c-Abl plus endogenous c-Abl) were calculated from their individual autoradiographic densities and normalized to the corresponding GFP-tagged protein level. All values obtained were then expressed as a percentage of the control value obtained from H₂O₂-untreated cells. Data are the mean +/− SEM of 3 independent experiments. Asterisks indicate statistical significance versus cells without H₂O₂. * P <0.05, **P <0.01.