PTEN regulates Rac activation and IS growth. (A) Left, OT-1 CTLs expressing shRNA specific for PTEN or a control shRNA (shNT) were stimulated with beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD, and activation of Rac was determined by immunoblot. Numbers denote corrected, normalized amounts of activated Rac. Right, CTL lysates were probed for PTEN by immunoblot. Numbers denote corrected, normalized amounts of PTEN. (B) OT-1 CTLs expressing shRNA against PTEN or nontargeting control shRNA were imaged on stimulatory bilayers after phalloidin staining. Left, representative TIRF images are shown. Right, quantification of cell spreading and F-actin clearance ratio (n = 35 cells). (C) Dock2^−/− 2B4 T cells expressing the indicated shRNAs were imaged on stimulatory bilayers as described in B. Left, representative TIRF images are shown. Right, quantification of cell spreading (n > 30 cells). 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. ***, P < 0.001 and ns, P > 0.05. Data are representative of at least two independent experiments.