Cdc42 and Rac1 are necessary for targeting Lck to the plasma membrane in Jurkat cells. (A) Jurkat cell extracts were loaded or not with GTP analog GMP-PNP and incubated with GST alone or fused to the indicated fragments of INF2. As controls, the Rho-GTPase binding domains of rhotekin (RBD) or PAK1 (PBD) were used to detect active RhoA or Rac1 and Cdc42, respectively. Ten percent of the original extract was immunoblotted in parallel. (B-C) Jurkat cells expressing GFP and shRNA control or shRNA targeted to RhoA, Cdc42, or Rac1 from the same plasmid were analyzed by immunoblotting with antibodies to the indicated proteins (B). As a loading control, the levels of GADPH were examined. Cells were also fixed, permeabilized, and stained for Lck (C). Double transfectants expressing shRac1 and shCdc42 from plasmids which coexpress GFP and Cherry, respectively, were also analyzed in panel C. (D) Jurkat cells expressing GFP fusions of Rac1V12, Cdc42V12, Rac1N17, or Cdc42N17 for 24 hours were stained for Lck. Anti-Lck antibodies and secondary antibodies coupled to Alexa-647 were used in panels C-D to detect Lck. (E) The percentage of cells with low levels of peripheral Lck in cells with silenced expression or overexpression of the indicated Rho-family GTPases was quantified and summarized as means ± SEM from 3 independent experiments. More than 50 transfected cells were scored in each experiment (**P < .01). (F) Jurkat cells transfected with GFP fusions of Rac1V14 or Cdc42V14 for 24 hours were stained for endogenous INF2 using anti-INF2 antibodies and secondary antibodies coupled to Alexa-594. The images shown were deconvoluted. The right panels show plots of colocalization of Rac1V14 or Cdc42V14 with INF2. The Pearson correlation coefficient (r) was 0.75 and 0.87 for Rac1V14/INF2 and Cdc42V14/INF2, respectively. Four independent experiments were performed. Scale bars indicate 5 μm.