Figure 2. AP/MS Identifies New KRAS Effectors Underlying RAS-Driven Macropinocytosis and Migration.

A Network diagram showing PPIs between RAS proteins, RIN1 and RADIL. B Schematic of assay to assess macropinocytosis (MPC), derived from (25). C MPC was quantified by TMR-dextran (red) signal under fluorescence microscopy. RAS inhibition reduced dextran uptake in wild-type, but not RIN1 knockout cells. Error bars indicate 95% confidence intervals estimated from 76 fields from the wild type and 226 from knockouts (Supplementary Methods). * : p<0.05 by two-sided Student’s t-test D Left: Superposition of crystal structures of the RA domain of RAF1 (PDB ID: 4G0N, brown) and RADIL (PDB ID: 3EC8, gray). RADIL R99 superimposes near RAF1 R89, which is required for RAS-RAF binding (20). E Cell-free co-immunoprecipitations indicated myc-tagged constructs as prey, and indicated GST-RAS fusion proteins as bait. myc-RADIL binds RAS directly, and its RA domain (residues 61–164) is sufficient. R99 is required. F Quantitative Real-Time PCR of 3 indicated siRNAs against RADIL (see Supplementary Methods). Total RNA was isolated from transfected H23 cells at 24, 48, and 72hrs post-transfection and the relative levels of RADIL transcript were quantified. For experiments with RADIL knockdown, a 1:1:1 pool of all three siRNAs were used. n=4 for all samples. G Schematic of scratch-wound assay. Cells transfected with anti-RADIL siRNA or a negative control were replated into a confluent monolayer. A wound was introduced in the monolayer, and the closure of the wound was monitored over time. H Quantification of cellular velocity into the wound over 24 hours; RADIL knockdown increased migration speed, but not when KRAS was inhibited. 5–6 wounds were quantified per condition, imaged hourly (Supplementary Methods). * p<0.05 by two-sided Student’s t-test.