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Sci. Signal., 28 July 2009
[DOI: 10.1126/scisignal.2000397]

Supplementary Materials for:

Cell Type–Specific Importance of Ras–c-Raf Complex Association Rate Constants for MAPK Signaling

Christina Kiel* and Luis Serrano

*To whom correspondence should be addressed. E-mail: christina.kiel{at}crg.es

This PDF file includes:

  • Materials and Methods
  • Fig. S1. Overview of EGF signal transduction.
  • Fig. S2. Comparing experimental and FoldX predicted kon and ΔG values.
  • Fig. S3. Predicted ΔGint values for Ras-Raf WT and mutant complexes as predicted by FoldX.
  • Fig. S4. ERK phosphorylation kinetics and Sos1 upshift in PC12 cells and mouse embryo fibroblasts.
  • Fig. S5. Dose-response curve of ERK-P 5 min after EGF stimulation over a concentration range of 0.075 to 50 ng/ml.
  • Fig. S6. Correlation of ERK phosphorylation and luciferase activity in RK13 cells.
  • Fig. S7. Effect of ERK inhibitor on luciferase activity, as a measure of ERK target gene–dependent gene activation, in RK13 cells.
  • Fig. S8. Transient expression of TAP (Flag-StrepII)-c-Raf plasmids.
  • Fig. S9. Luciferase assay and ERK-P kinetics for c-Raf WT, A85K, and R89L in RK13 and HEK293 cells.
  • Fig. S10. Abundance of c-Raf WT and mutants in RK13 and HEK293 cells.
  • Fig. S11. PI3K contribution to ERK phosphorylation at different EGF concentrations in RK13 cells.
  • Fig. S12. PI3K contribution to ERK phosphorylation in HEK293 cells.
  • Fig. S13. Luciferase activities for c-Raf WT, A85K, and R89L in HEK293 and RK13 cells treated with the PI3K inhibitors LY294002 or wortmannin.
  • Fig. S14. Comparing experimental and simulated time courses for Sos1, EGFR-P, and ERK-P in HEK293 cells and HEK293-like model.
  • Fig. S15. Comparing experimental and simulated time courses for EGFR-P and ERK-P in RK13 cells and RK13-like model.
  • Table S1. Comparing FoldX mutagenesis results and ionic strength dependence of WT with in vitro experiments using stopped-flow and isothermal titration calorimetry.
  • Table S2. Design of c-Raf mutants using FoldX.
  • Table S3. Modeling parameters.
  • Appendix: Sensitivity Analysis

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Technical Details

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Citation: C. Kiel, L. Serrano, Cell Type–Specific Importance of Ras–c-Raf Complex Association Rate Constants for MAPK Signaling. Sci. Signal. 2, ra38 (2009).

© 2009 American Association for the Advancement of Science

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