Inhibition of TCR-dependent Vav phosphorylation and downstream signaling by NSAIDs. (A) Immunoblot analysis using antiphosphotyrosine mAb of Vav-specific immunoprecipitates from Jurkat cells, nonactivated (0) or activated by CD3 ligation (CD3) for 1 minute, in the presence or absence of 600 μM ibuprofen (ibupr), 15 μM sc-560, or 250 μM NS-398. The filter was stripped and reprobed with anti-Vav mAb. (B) Immunoblot analysis with anti-Rac mAb of in vitro binding assays of postnuclear supernatants from Jurkat cells, using an agarose-conjugated Pak1-GST fusion protein. Equal amounts of postnuclear supernatants from the same samples were separated on the same gel (bottom). Cells were either nonactivated (0) or activated by cross-linking of CD3 (1 minute and 5 minutes) in the presence or absence of 15 μM sc-560. (C) Immunoblot analysis of postnuclear supernatants from Jurkat cells, nonactivated (0) or activated by CD3 ligation as in panel B (1 minute CD3 cross-linking). The filter was probed with antiphospho–Pak1 antibody, followed by antiactin mAb as loading control. The migration of molecular mass markers is shown.