Fig. 1. NRas and BRAF reside and associate in clusters at the plasma membrane of melanoma cells that are altered after EGF activation.

(A) Two-colour PALM imaging of resting 108T melanoma cells expressing PAmCherryNRas and PAGFP-BRAF. Cells were seeded on the coverslip for 2 days before fixation. Shown is a representative cell (N=20). Bars – 2 μm (left) and 200 nm (right). (B) PCF of PAGFP-BRAF. (C) PCF of PAmCherry-NRas. (D) The density of BRAF and NRas at the PM. (E) A two dimensional map of self-clustering (value of g(0-100)) vs. protein density. Values are shown for individual cells as discs, either for BRAF (green) or NRas (red). Note that both axes are logarithmic. Dashed black lines are guidelines for comparison of results with additional measurements. (F) Bivariate PCF (BPCF) of BRAF and NRas (representative data shown for a single cell; N=20). (G) The extent of mixing (EOM) of BRAF and NRas (averaged over 20 cells) (See Analyses for further details). (H) Twocolour PALM imaging of 108T melanoma cells expressing PAmCherry-NRas and PAGFPBRAF. Cells were seeded on the coverslip for 2 days, and activated with EGF before fixation. Shown is a representative cell (N=27). Bars – 2 μm (left) and 200 nm (right). (I) PCF of PAGFP-BRAF. (J) PCF of PAmCherry-NRas. (K) The density of BRAF and NRas at the PM. (L) A two dimensional map of self-clustering (value of g(0)) vs. protein density. Data presentation is as in panel E. (M) Bivariate PCF (BPCF) of BRAF and NRas. (N) The EOM of BRAF and NRas. (O) Cumulative distributions of cluster sizes of (left) BRAF or (right) NRas. The distributions are presented in copy numbers, as quantified by a clustering algorithm applied on the PALM images (see Methods). Results are shown for resting and EGF-activated 108T cells. P-values relative to data in Fig. 1E: * < 0.05, ** < 0.01, *** < 0.005. P-values are summarized in Supplementary Table S4. Error bars in all relevant panels are SEM due to measurements on multiple cells.