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. 2015 Aug 11;5:688–704. doi: 10.1016/j.fob.2015.08.005

Fig. 6.

Fig. 6

Defective TCR-induced signaling in the absence of GIT2. Purified splenic T cells from the Git2+/+ and Git2−/− mice were stimulated by 5 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 at indicated time. (A) ERK1/2 and PAK phosphorylation were analyzed using phospho-specific antibodies. Nonphosphorylated proteins served as a control. The immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (bottom). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05). (B) Cell extracts were incubated with glutathione S-transferase-PAK RBD to precipitate GTP-bound Rac1 (Rac1-GTP). Anti-Rac1 antibody was used to detect GTP-bound GTPases. Total Rac1 served as a control. The immunoblot bands were scanned for densitometry analysis with the value obtained from control cells set as 1 (right). The results were representative of three independent experiments and error bars represent standard deviations (*P < 0.05). (C and D) The cells were fixed, stained with FITC-phalloidin and anti-CD3, and analyzed by confocal and FACS. All data are representative of at least three independent experiments, Scale bar, 10 μm. All data are representative of at least three independent experiments.