Fig. S5

Model Fitting, In-Cell Control Validation, and Results, Related to Figure 4

(A) 3T3 cells expressing JNK KTR nonphosphorylatable (AA) and phosphomimetic (EE) mutants were imaged upon addition of Leptomycin B (50 ng/ml). Images were quantified as descrived in Methods. Data represent the mean ± SD of more than 100 cells (Data) obtained from 3 independent experiments. Data were fitted to the model obtaining rate constants for each individual cell. Mean ± SD of simulated data is shown (Model).

(B) Correlations of estimated import and export rate constants for JNK KTR AA and EE obtained in Panel a.

(C) Cells expressing indicated mutant versions of JNK KTR were imaged and quantified under basal conditions. Histograms of basal C/N ratio are shown. Solid gray lines are probability densities of the same log-normal distribution (? = 0.02, ? = 0.22) that has been shifted to fit each mutant.

(D and E) Cells expressing two versions of JNK KTR (wild-type and AE [D] or AE and EA [E]) were imaged at a single time point and quantified as described in Methods. Values of C/N for wild-type and mutant JNK KTRs were obtained. Correlations for each individual cell between mutants AE versus EA (E) and WT versus AE (D) are shown. Data represent more than 100 cells.

(F) Schematic representation of the temporal profile assumed for estimating kinase activity. Four times (T1-T4) and 3 concentrations (C1-C3) were explored to estimate the kinase activity dynamics that generate the observed KTR dynamics.

(G and H) distributions of the parameters shown in Panel b for cells stimulated with 1 ng/ml IL-1?. Individual data points (blue dots), mean (red line), 95% SEM interval (red shadow) and SD (blue shadow) are shown for each parameter. Data represent 302 cells.

(I and J) C/N ratios were obtained from the temporal profiles calculated for Figure 4E (Initial). To test the robustness of our fitting procedure, noised ratio dynamics were then used to recalculate kinase activity temporal profiles (Final). Correlations between initial and final maximum active JNK concentration (I) and area under the curve (AUC) (J) are shown.

(K and L) Model based relationship between steady state C/N ratio (K) or half response time (L) and concentration of active kinase when kinase is localized just in the Cytoplasm (green), Nucleus (red) or Both (blue). JNK KTR parameters were used.