Fig. 3
R-RAS2 interacts with plasma membrane receptors known to be important for breast cancer. a, Selected list of plasma membrane proteins that interact with R-RAS2 in a murine breast cancer tumor isolated from a Rosa26-RRAS2fl/fl x Wap-Cre female mouse. Full data can be found in Extended Data Table 6. The known involvement of these proteins in cancer and breast cancer is briefly summarized, and the Mascot Score for the specific affinity column with anti-hemagglutinin (for hemagglutinin-tagged R-RAS2) and a control isotypic immunoglobulin column is also shown. Protein function and association with cancer data have been retrieved from The Human Protein Atlas: https://www.proteinatlas.org. b, Biological processes and pathways found to be significantly altered after KEGG analysis of the R-RAS2 plasma membrane interactome of Extended Data Table 6. The x-axis shows the percentage or proteins in the pathway found associated to R-RAS2 and the y-axis the–Log10 of the P adjusted value. Full data is provided in Extended Data Table 7. c, STRING network of physical and functional interactions among the indicated proteins from Fig. 3a. Pink lines indicate experimentally-determined interactions; blue lines indicated interactions found in curated databases; green lines indicate interactions found by textmining and black lines indicate co-expression. d, Co-immunoprecipitation of CD44, Epha2 and Slc3a2 (CD98hc) with R-RRAS2 (IP anti-Hag) was studied in detergent lysates of the CBM-MBC21 mouse cell line. The membranes were re-probed with anti-Hag as a control for loading of R-RAS2, and the arrows indicate the positions of the co-immunoprecipitated proteins and those in the whole cell lysate (WCL). The molecular weight markers are shown on the left. e, Co-immunoprecipitation of CD44, Epha2 and Slc3a2 (CD98hc) with R-RRAS2 in detergent lysates of the human breast cancer cell line BT-549 transfected with a Hag-tagged R-RAS2 construct. Legend as in panel d. f, Mid-plane confocal microscopy sections of CBM-MBC21 cells fixed and stained with R-RAS2 (anti-Hag) and anti-CD44 antibodies to show their co-localization at the plasma membrane. The nucleus of the cells is stained with DAPI (blue). Details of 4 areas of the plasma membranes are shown to the right to show the co-localization of R-RAS2 with CD44 in cell protrusions. g, Co-localization of R-RAS2 and CD44 was measured by analysis of all pixels in 23 cell protusions as in the inset of Fig. 3f and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median (= 0.70) and the 75% and 25% percentiles. h, Mid-plane confocal microscopy sections of CBM-MBC21 cells fixed and stained with R-RAS2 (anti-Hag) and anti-CD98hc antibodies to show their co-localization at the plasma membrane. The nucleus of the cells is stained with DAPI (blue). Details of 4 areas of the plasma membranes are shown to the right to show the co-localization of R-RAS2 with CD98hc at the external (apical) plasma membrane of the cell aggregate. i, Co-localization of R-RAS2 and CD98hc was measured by analysis of all pixels in 25 membrane regions as in the inset of Fig. 3h and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median (= 0.69) and the 75% and 25% percentiles