Figure 5.

CD6 associates to SLP-76 after TCR engagement. (a) CD4⁺ T cells from wild-type (WT) or Slp76^OST mice were left unstimulated (0) or stimulated for 30, 120 and 300 s with pervanadate. Cell lysates were directly processed for immunoblot (Total lysates: TL) or subjected to affinity purification on Strep-Tactin-Sepharose beads (Affinity purification: AP). Equal amount of lysates and eluates were analyzed by immunoblot with anti-CD6, anti-SLP-76 and anti-GADS. (b) CD4⁺ T cells from wild-type (WT) or Slp76^OST mice were left unstimulated or stimulated for 30 and 120 s with anti-CD3 and anti-CD4. Cell lysates were directly processed for immunoblot (Total lysates) or subjected to affinity purification (AP) as described in (a). Equal amount of lysates or eluates were analyzed by immunoblot with anti-CD6 or anti-SLP-76. (c) CD4⁺ T cells from wild-type mice were left untreated (-) or stimulated for 2 min with anti-CD3 and anti-CD4 (+). Total lysates (TL) were directly analyzed by immunoblot (IB) or subjected to immunoprecipitation (IP) with an anti-CD6 (Anti-CD6 IP). Total lysates and anti-CD6 immunoprecipitates were analyzed by immunoblot (IB) with anti-CD6 or anti-SLP-76. (d) CD4⁺ T cells from wild-type mice were treated as in c. Total lysates were immunoprecipitated (IP) with an anti-CD6 (CD6) or an isotype control antibody (iso). Total lysates and anti-CD6 immunoprecipitates were analyzed by immunoblot with anti-phosphotyrosine (Anti-p-Tyr) or anti-CD6. (e). CD4⁺ T cells from wild-type mice were treated as in (c). Total lysates were immunoprecipitated with anti-Vav1. Total lysates(TL) and anti-Vav1 immunoprecipitates (IP) were analyzed by immunoblot (IB) with anti-Vav1 or anti-CD6. Data in (a) to (e) are representative of at least two independent experiments.