Fluorescent GEF activity measurements in vitro Nucleotide loading and nucleotide exchange measurements on Ras were performed as described previously in Margarit et al., 2003 [43]. Dissociation rates were measured on a fluorescence spectrometer combined with a stop-flow device (SFM-400; Bio- Logic). Substrate solution containing 1 mM mantGDP-Ras was automatically mixed with reaction buffer supplemented with 200 mM GDP and additional proteins such as SOS and/or ezrin, when indicated. Dissociation was measured for 360 s. The data were fitted either to a linear (Y = I+B*x, with nucleotide dissociation rate = absolute(B)), single exponential (Y = Y0+ A12k1*x) or bi-exponential (Y = Y0+A12k1*x+A22k2*x) function (with I as the intercept, B as slope, A1/A2 as the amplitude, Y0 as the fluorescent value at infinite time, k1/k2 as the apparent dissociation rate constant and x as time) using the program Origin 8Pro. Supporting Information Figure S1 ERM proteins are necessary for PDGFR signaling. A, Loss of ERM binding to CD44 tail upon point mutation of CD44. CD44 tail fused to GST was over-expressed in RT4 cells kept at low cell density. Cells were lysed, GST-CD44 tail was affinity-precipitated and derived complexes were analyzed by Western blot employing a mixtureof antibodies against ERM proteins and GST for loading. The mutated CD44 tail reveals strongly diminished though not absent ERM binding. B–D, Removal of the ERM proteins from the co-receptor at the plasma membrane interfered with signalling. RT4 cells were transfected with a construct expressing either the soluble ezrin-binding domain of CD44 (GST-CD44tail) or the point-mutated domain defective in ezrin binding (GST-CD44tail mutant). Transfected cells were plated at low density,serum starved overnight prior to induction with PDGF (10 ng/mL), lysed and immunoblotted as indicated. 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F, HeLa cells were plated at low density, treated with a cocktail of siRNAs against all three ERM proteins or control siRNA for 24 h, then serum starved overnight prior to treatment with PDGF (10 ng/ml, 5 min). Lysates immunoblotted as indicated. (TIF) Figure S2 Ezrin R579A mutant inhibits PDGF-induced Erk phosphorylation. A, RT4 cells expressing dox-inducible ezrin wildtype-VSVg were plated at low density, serum starved overnight prior to treatment with either PDGF (20 mM, 5 min), TPA (100 ng/ml, 5 min) or IL-6 (1 ng/ml, 5 min). Lysates immunoblotted as indicated. B, RT4 cells expressing dox- inducible ezrin R579A mutant-VSVg were plated at low density, serum starved overnight prior to treatment with either PDGF (20 mM, 5 min), TPA (100 ng/ml, 5 min) or IL-6 (1 ng/ml, 5 min). Lysates immunoblotted as indicated. (TIF) Acknowledgments We appreciate Alfred Wittinghofer’s generosity concerning plasmids and proteins. 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Vetrivel KS, Cheng H, Kim SH, Chen Y, Barnes NY, et al. (2005) Spatial segregation of gamma-secretase and substrates in distinct membrane domains. J Biol Chem 280: 25892–25900. PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27511