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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 5: — C-terminus and N-terminus of G3BP1 required for cell extension formation. (A) Cells transfected with HA-tagged G3BP1 were plated on collagen, and pull-down assays were performed with glutathione beads bound G3BP1 peptides (GST-G3BP1 [FL], GST-G3BP1 amino acids 1–236 [N-terminus], GST-G3BP1 amino acids 230–466 [CT]) and HA-tagged R-ras detected by running the bound proteins on a Western and identified by probing with HA antibody. The results show that the interaction between the R-ras maps to the C-terminal domain of G3BP1 (amino acids 230–466) and full-length G3BP1 and weak interactions detected with N-terminal domain (amino acids 1–236). Blots shown from three experiments. (B) Cells transfected with HA-tagged G3BP1 truncated constructs (FL, NT, and CT) were immunoprecipitated with anti-R-ras and immunoprecipitates separated by Western blotting probed with HA antibody to determine the association of R-ras with specific domain of G3BP1. Experiment repeated three times. (Ci–Ciii) FliI KO fibroblasts transfected with either FliI-GLD, FliI-LRR regions, or empty construct were cotransfected with HA-tagged truncated G3BP1 fusions. Transfected cells plated on collagen and lysates subjected to immunoprecipitation with HA antibody determined the relationship between R-ras and HA-G3BP1 fusion domains in FliI knockout fibroblasts expressing FliI-GLD and FliI LRR domains. These experiments show association between R-ras and full-length G3BP1 and C-terminal domain of G3BP1 in FliI-LRR and weak association in FliI-GLD cells or cells with empty construct. Experiments repeated three times. (Di) Confocal images of HA-tagged G3BP1 (FL, NT, and CT) fusion proteins transfected cells plated on collagen. (Dii) Histogram shows 2.5-fold increase (p < 0.05) in mean number and length of cell extensions in HA-G3BP1 full-length-transfected cells and not in N- or C-terminal truncated domains, suggesting, probably, that G3BP1 interactions at the C-terminal are also required for FliI to regulate cell extension formation. Data reported as mean ± SD and analyzed by ANOVA. (E) Cells cotransfected with Rasgap or control siRNA and HA-tagged G3BP1 that were plated on collagen show cell extensions in transfected (arrows) and nontransfected cells. Observations recorded in 30 cells. (F) Pull-down assay showing association of endogenous Rasgap¹²⁰ with N-terminus of G3BP1 and weak association with C-terminus.