FIG. 3.

Activation of Rap1 requires PLD activity. (A) Jurkat T cells were serum starved for 2 h and treated with or without t-butanol or n-butanol for 15 min prior to stimulation with anti-CD3 antibodies for 10 min, as indicated. Cell lysates were immunoblotted for total Rap1 and GTP-Rap1 (GST-RalGDS-RBD pulldown assay). (B) Jurkat T cells expressing YFP-RalGDS-RBD, a probe for GTP-bound Rap1, and the indicated Rap1 construct were imaged live growing in serum. (C) Serum-starved Jurkat T cells expressing YFP-RalGDS-RBD and Rap1 were treated with or without t-butanol or n-butanol and imaged live before and after stimulation with anti-CD3 antibodies. Bars, 5 μm. (D) Quantification of the results shown in panel C, plotted as means± SEM (n = 4).