Fig. 2: SLC3A2 associates with RAS and controls mTORC1 activity.
a Co-immunoprecipitation of SLC3A2 with mutant isoforms of mNeonGreen-tagged KRAS and NRAS. mNeonGreen-tagged KRASG12D was used in RPMI 8226 and XG2, NRASG12D in SKMM1 and NRASQ61L in L363. Representative blots; n = 4. b Essential interactome of SLC3A2 in RPMI 8226. CRISPR screen score (CSS; x-axis) plotted by the BioID2-SLC3A2/empty vector enrichment (y-axis). Essential interactome is labeled in pink (≤−1.0 CSS and ≥1.0 log2fc BioID2-SLC3A2). c Proximity ligation assay (PLA) (red) of SLC3A2 and RAS in RPMI 8226 and SKMM1 cells. Cells were counterstained with DAPI (blue) and wheat germ agglutinin (WGA; green). Scale bar is 10 μm, representative images; n = 3. d PLA score of cells transduced with indicated shRNAs following puromycin selection for two days. Data from three independent experiments for KRAS, NRAS and SLC3A2 knockdown, and 2 independent experiments for SLC7A5 knockdown. Data pooled from independent experiments; the number of cells quantified per condition is listed in the source data file. *** denotes P value < 0.0001 by one-way ANOVA with Dunnett’s post test; box plots represent median and 25–75% of data, whiskers incorporate 10–90% of data, and outliers are displayed as dots. e Global changes in phosphorylation measured by quantitative MS following knockdown of SLC3A2 in SKMM1. Change in phosphorylation (log2fc) in cells expressing shSLC3A2 vs. shCTRL (x-axis) plotted by the measured intensity (y-axis). f Identification of genes that regulate phosphorylation of RPS6 at S240/244 by CRISPR screening. Averaged CRISPR screen results from sorted RPMI 8226 and SKMM1 cells comparing cells with low p-RPS6 vs. high p-RPS signal. All values are averaged for both cell lines except for KRAS and NRAS, which are shown for RPMI 8226 and SKMM1, respectively. Source data are provided as a Source Data file.
