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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Nat Immunol. 2014 Mar 2;15(4):384–392. doi: 10.1038/ni.2843

Figure 6.

Figure 6

SLP-76 associates with CD6 in a Lat-independent manner. (a,b) Jurkat cells (clone E6.1) and their Zap70-deficient (P116) and Lat-deficient (JCam2.5) variants were transfected to co express CD25ξ and human CD6. The resulting E6.1-CD25ξ-CD6 and P116-CD25ξ–CD6 cells were left unstimulated (-) or were stimulated (+) for 2 min with anti-CD25. Total lysates (a) or anti-CD6 immunoprecipitates (b) were analyzed by immunoblot with anti-phosphotyrosine (Anti-p-Tyr), anti-Zap70 and anti-CD6. (c) E6.1-CD25ξ-CD6 and JCam2.5-CD25ξ-CD6 were stimulated as in (a). Total lysates were analyzed by immunoblots with anti-CD6, anti-phosphotyrosine (Anti-p-Tyr), anti-SLP-76 and anti-LAT. (d) E6.1-CD25ξ-CD6 and JCam2.5-CD25ξ-CD6 cells were stimulated as in (a) and total lysates were subjected to immunoprecipitation with an anti-SLP-76. Immunoprecipitates (IP) were analyzed by immunoblots with anti-CD6, anti-phosphotyrosine (Anti-p-Tyr), anti-SLP-76 and anti-LAT. (e) CD4+ T cells made defective in Lat (LatΔ/Δ; see Online Methods) and wild-type CD4+ T cells (Lat+/+) were stimulated with anti-CD3 and anti-CD4 for 2 min at 37 °C. Total lysates(TL) and anti-SLP-76 immunoprecipitates (IP: anti-SLP-76) were analyzed by immunoblots (IB) with anti-CD6, anti-SLP-76 and anti-LAT. (f) Lat-deficient, JCam2.5-CD25ξ cells lacking CD6 (CD6−) or expressing CD6 (CD6+) were left unstimulated (-) or were stimulated (+) for 2 min with anti-CD25. Total lysates were analyzed by immunoblot with anti-phosphotyrosine (Anti-p-Tyr), anti-SLP-76 and anti-CD6. Data are representative of at least two independent experiments.