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. 2018 Oct 1;29(20):2481–2493. doi: 10.1091/mbc.E18-03-0147

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© 2018 Arora et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — FliI needed for the assembly of R-ras/G3BP1. (A) FliI WT and KND cells show equivalent expression levels of G3BP1 protein. (B) Pull-down assay shows proteins eluted from GST only, GST RasG15A, and GST Ras WT Sepharose beads incubated with cell lysates. There are increased levels of G3BP1 protein on GST Ras G15A beads. Equal amounts of proteins loaded on beads (INPUT). These experiments repeated four times. (Ci) Knockdown of G3BP1 expression with two different sets of siRNA showing 75 and 72% decrease in protein levels. (Cii, Ciii) Confocal images of FliI WT and KND cells transiently cotransfected with G3BP1 siRNA and fluorescent siRNA (siGloDY-547) show a 2.5-fold difference in development of cell extensions in nontransfected FliI WT and FliI KND cells (p < 0.05). In G3BP1 siRNA-transfected cells there was a sevenfold reduction in FliI WT (p < 0.05) cells and 2.4-fold reduction in FliI KND (p < 0.05) as compared with nontransfected cells. Data in histogram are from three different experiments. Data reported as mean ± SD, analyzed by ANOVA (p < 0.05). (D) Confocal optical sections showing localization of G3BP1 and GFP-FliI to the adhesions sites on collagen substrate. Observations recorded in 25 cells. (E) HA antibody immunoprecipitation of cells transfected with HA-tagged full-length FliI (FL), HA-GLD domain, and HA-LRR domain show association of FliI FL and FliI LRR to G3BP1. Experiment repeated three times. (F) Confocal images showing colocalization of G3BP1 and R-ras at the cell extensions. Observations recorded in 30 cells. (G) Immunoprecipitaion with HA antibody of cells transfected with HA-tagged or control, HA-untagged CA R-ras, and DN-R-ras show interaction between G3BP1 and CA R-ras. (H) Cells transfected with HA-tagged CA-R-ras, CA-R-ras MUT (P202A; P203A), and WT R-ras plated on collagen or in suspension. Cell lysates immunoprecipitated with HA antibody and analyzed by SDS–PAGE gels showing interaction between constitutively active-R-ras and G3BP1 and not between mutant R-ras and G3BP1 or cells in suspension (sus). (I) FliI WT and KND cells were transfected with HA-tagged CA R-ras. Lysates from cells plated on collagen immunoprecipitated with HA antibody showed absence of FliI reduced association between G3BP1 and R-ras. Experiment in G, H, and I repeated three times.