211 to 0. 5, 1, 2 and 4. then The model was simulated for 2880 minutes for each perturbation and the resulting MITF activity was compared for the four PIAS3 levels. The success criterion used in the sensitiv ity analysis was that MITF activity should be monotoni cally decreasing with increasing PIAS3 and that the MITF activity Inhibitors,Modulators,Libraries at the highest PIAS3 amount should be less than the half that of the lowest PIAS3 amount. In experiments 6 and 7, we have mimicked the experiments presented in, Figure 2. Here the authors have investigated the association between MITF and PIAS3 in BL6 B16 cells before and after 10 and 30 minutes of activation with c kit ligand. In experiment 6 only GFP PIAS3 was transfected, while in experiment 7, the cells were transfected with both MITF and PIAS3.

In experiment 6, activation is performed by increase of the levels Inhibitors,Modulators,Libraries of phosphorylated ERK and JAK from 10 to 500 and 50, respectively. In experiment 7, transfection was simulated by elevation of the MITF production rate from 1 to 10 and the PIAS3 production rate from 1. 262 to 10 and simulated for 2880 minutes. Thereafter, activation was simulated by increase of the amount of phosphorylated ERK and JAK from 10 to 1200 and Inhibitors,Modulators,Libraries 250, respectively. The success criterion used for the sensitivity analysis in both experiments was that the total amount of MITF PIAS3 complex should be higher after 10 minutes of stimulation compared to before stimulation and that the amount of complex should be lower after 30 minutes of stimulation than after Inhibitors,Modulators,Libraries 10 minutes. Experiment 8 simulated the experiment from, Figure 3A, left.

The authors co transfected NIH 3T3 cells using MITF, with a constitutively active RSK1 plas mid and with a reporter gene to read MITF activity. To simulate transfection Inhibitors,Modulators,Libraries of MITF, the MITF production rate was elevated from 1 to 7. The amount of RSK1 is not dynamically determined in the model, and thus the transfection of this product is approximated with an ele vation of the static amount from 500 to 5000. The manipulated model was simulated for 2880 minutes, and the MITF activities with and without RSK1 were compared. The success criteria used in the sensi tivity analysis was that the two cases should not differ more than twofold. Experiments 9 to 12 represent simulations of the experiment presented in Figure 3B.

Here, the authors transfected NIH 3T3 cells with MITF or the MITF S409A mutant, with constitutively active RSK1 and with a reporter gene to monitor MITF transcrip tional activity. To simulate the transfection of MITF, the MITF production rate was elevated from 1 to 7. To simulate the transfection of PIAS3, the PIAS3 produc tion rate was elevated from 1. 262 to 4. sellectchem The amount of RSK1 is not dynamically determined in the model, and thus the transfection of this product is approximated with an elevation of the static amount from 500 to 5000.