Extended Data Fig. 2: Plasma membrane-localized RAC1 is required for RAS-induced macropinocytosis.
From: Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis
a, b, Effect of V1A expression on RAC1 activity. a, Immunoblots of RAC1 wild type and RAC1(L61) activity (GST–PBD, pull-down of GFP–RAC1–GTP; vinculin loading control) with or without V1A knockdown in HeLa HRAS(G12V) cells (Ctl, GFP). b, Immunoblot of endogenous RAC1 activity (GST–PBD, pull-down of RAC1–GTP; tubulin loading control) in HeLa Ctl and HRAS(G12V) cells with or without V1A knockdown. c, Effect of V-ATPase depletion (siV1A) and rescue by plasma membrane-targeted RAC1 (GFP–RAC1(L61) K-tail) on RAC1(L61) localization and macropinocytosis in HeLa HRAS(G12V) cells. Fluorescence micrographs of GFP–RAC1 (top), TMR–dextran uptake (bottom) and quantification of TMR–dextran (bar graph). d, e, Effect of V-ATPase or RAC1 depletion on macropinocytosis. Quantification of TMR–dextran uptake following bafilomycin A1 treatment (d) or RAC1 knockdown (e) in the absence or presence of exogenous cholesterol in HeLa HRAS(G12V) cells. f, Fluorescence micrographs of cholesterol localization (filipin, top), GFP–RAC1 localization (middle) and TMR–dextran uptake (bottom) in V-ATPase-depleted HeLa KRAS(G12V) cells in the absence or presence of exogenous cholesterol. g, Quantification of TMR–dextran uptake in mutant RAS cells with V1A knockdown and rescue by plasma membrane-targeted RAC1 (GFP–RAC1(L61) K-tail). Images (c, f) and immunoblots (a, b) are representative of three biological replicates. Data in c are mean ± s.e.m. representing the fraction of cells that display plasma membrane localization of GFP–RAC1(L61) or GFP–RAC1(L61) K-tail. In f, the dashed lines delineate the cell and nucleus and data are mean ± s.e.m. representing the fraction of cells that display plasma membrane localization of cholesterol (top) or GFP–RAC1 (middle). Scale bars, 10 μm. At least 500 (c–g) cells were quantified in each biological replicate (n = 3); data are mean ± s.e.m.; unpaired two-tailed Student’s t-test. Gel source data for a, b are shown in Supplementary Fig. 1.
