Figure 10.
Figure 10. Ras drives PI3K activation and F-actin remodeling at the IS. (A and B) OT-1 CTLs expressing GFP-labeled V12Ras or GFP alone were stimulated on lipid bilayers containing pMHC and ICAM-1 in the presence of either wortmannin (Wort; A) or U0126 (B) as indicated (Veh = vehicle control). Cells were then fixed and phalloidin-stained. Quantification of cell spreading is shown (n ≥ 30 cells). Representative TIRF images of V12Ras-expressing cells and GFP-expressing controls are shown to the left in A. (C) OT-1 CTLs were transduced with nontargeting control shRNA (shNT) or shRNA specific for Nras either in the presence or the absence of shRNA-resistant, GFP-labeled Nras. Cells were then stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with phalloidin. Left, representative TIRF images are shown. Middle, quantification of cell spreading and F-actin clearance ratio (n ≥ 37 cells). Right, Nras protein expression by immunoblot. Numbers denote corrected, normalized amounts of Nras. (D) OT-1 CTLs expressing nontargeting control shRNA (shNT) or shRNA specific for Kras were stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with phalloidin. Left, quantification of cell spreading and F-actin clearance ratio (n = 30 cells). Right, Kras protein expression by immunoblot. Numbers denote corrected, normalized amounts of Kras. (E) OT-1 CTLs expressing V12ras (left) or shNras (right) were stimulated, along with the indicated controls, using beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD, and Rac activation was analyzed by immunoblot. Numbers denote corrected, normalized amounts of activated Rac. Bars, 5 µm. In scatter plots, red lines and error bars denote the mean and SEM, respectively. P-values were calculated using the Mann-Whitney test (two-tailed). ***, P < 0.001, **, P < 0.01, *, P ≤ 0.05, and ns, P > 0.05. Data are representative of at least two independent experiments.

Ras drives PI3K activation and F-actin remodeling at the IS. (A and B) OT-1 CTLs expressing GFP-labeled V12Ras or GFP alone were stimulated on lipid bilayers containing pMHC and ICAM-1 in the presence of either wortmannin (Wort; A) or U0126 (B) as indicated (Veh = vehicle control). Cells were then fixed and phalloidin-stained. Quantification of cell spreading is shown (n ≥ 30 cells). Representative TIRF images of V12Ras-expressing cells and GFP-expressing controls are shown to the left in A. (C) OT-1 CTLs were transduced with nontargeting control shRNA (shNT) or shRNA specific for Nras either in the presence or the absence of shRNA-resistant, GFP-labeled Nras. Cells were then stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with phalloidin. Left, representative TIRF images are shown. Middle, quantification of cell spreading and F-actin clearance ratio (n ≥ 37 cells). Right, Nras protein expression by immunoblot. Numbers denote corrected, normalized amounts of Nras. (D) OT-1 CTLs expressing nontargeting control shRNA (shNT) or shRNA specific for Kras were stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with phalloidin. Left, quantification of cell spreading and F-actin clearance ratio (n = 30 cells). Right, Kras protein expression by immunoblot. Numbers denote corrected, normalized amounts of Kras. (E) OT-1 CTLs expressing V12ras (left) or shNras (right) were stimulated, along with the indicated controls, using beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD, and Rac activation was analyzed by immunoblot. Numbers denote corrected, normalized amounts of activated Rac. Bars, 5 µm. In scatter plots, red lines and error bars denote the mean and SEM, respectively. P-values were calculated using the Mann-Whitney test (two-tailed). ***, P < 0.001, **, P < 0.01, *, P ≤ 0.05, and ns, P > 0.05. Data are representative of at least two independent experiments.

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