Extended Data Fig. 2: N-terminal disordered sequence of ARAF drives PM clustering.

a, 293FT cells transfected with indicated constructs were subjected for active RAS pulldown and western blot analysis. b, SKBR3 cells were transfected with indicated plasmids. Images were taken (left), percentages of cells with PM signals were quantified, n = 3 (right). Scale bar, 5 μm. c, Schematic representation of ARAF mutations identified in human cancers from cBioportal. d, 293FT cells transfected with NRAS and indicated constructs were collected and NRAS was immunoprecipitated. e, SKBR3 transfected with ARAF 255-GFP were stained with CellMask PM dye. Scale bars, 5 μm. f, ARAF 255 granule persistence after 5 minutes of 5% hexanediol treatment. Images were taken (left), percentages of remaining puncta signals were quantified, n = 5 (right). Scale bar, 5 μm. g, SKBR3 cells transfected with indicated plasmids were subjected for imaging (left), percentages of cells with PM signals were quantified, n = 3 (right). Scale bar, 5 μm. h, Immunofluorescent staining in HeLa and MCF7 cells. Scale bar, 5 μm. i, PM sub-localization of signaling components in SKBR3 cells were examined by western blot. j, ARAF or ARAF 255 granule persistence after 24 hours lapatinib or SHP099 treatment. Images were taken (left), percentages of remaining puncta signals were quantified, n = 3 (right). Scale bar, 5 μm. Immunoblots are representative of three independent experiments in a, d, i. Error bars denote the mean ± SD (biological replicates), and statistical analyses were performed using unpaired two-tailed Student’s t-test in b, f, g, j.

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