DT-2 sensitive, PKGI activity in RN46A cells. A) DEAE chromatography and phosphorylation assays of RN46A cell extracts were performed as described in Methods. Chromatogram displays cGMP-dependent (background-subtracted) kinase activity and quantitative protein measurement. cGMP-independent kinase activity was measured as 27.3% of total kinase activity. Representative immunoblot of fractions with detectable kinase activity is shown below chromatogram. B) Evaluation of DT-2 sensitivity in fractions containing detectable (13–17) or nondetectable (18–22) PKGI protein by immunoblot. Both fraction bins 13–17 and 18–22 displayed DT-2-sensitive kinase activity (***, p < 0.001; **, p < 0.01), with fractions overlaying PKGI protein being significantly more sensitive to DT-2 (***, p < 0.001). Dashed line represents cGMP-independent kinase activity equivalent to 27.3%. Data shown are from a single experiment, are representative of three independent experiments, and are presented as means ± SEM. Statistical significance was calculated via two-way ANOVA, followed by Bonferroni's post-test.