FIGURE 7.

NaPP1 selectively inhibits Hck-T338A in K562 cells. K562-Neo, K562-Hck, and K562-Hck-T338A cell populations were grown in 0.5% FBS overnight and treated with the indicated concentrations of imatinib and NaPP1 (3 μm) for 5 h. Hck was immunoprecipitated from clarified cell lysates and immunoblotted with a phosphospecific antibody that recognizes the tyrosine-phosphorylated activation loop of Hck (pHck). Duplicate membranes were blotted for Hck as a loading control. Western blots were analyzed using the Odyssey Infrared Imaging System. The Hck phosphotyrosine signal intensities were normalized to the levels of Hck protein from the blots of two independent experiments, and the average intensity ratios ±S.D. are presented in the bar graph. The entire experiment was repeated twice from independently derived cell populations with comparable results; representative blots are shown at the top. Two-way ANOVA was performed to determine statistical significance of the effect of imatinib and NaPP1 on the phosphorylation of Hck within each cell line. For K562-Hck, neither imatinib nor NaPP1 had a significant effect (p > 0.3), whereas for K562-Hck-T338A, NaPP1 treatment produced a significant reduction in Hck phosphorylation (p < 0.0001).