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. Author manuscript; available in PMC: 2012 Jul 15.
Published in final edited form as: J Immunol. 2011 Jun 17;187(2):805–816. doi: 10.4049/jimmunol.1003314

Figure 8. GADS and ITK expression and SLP76 activation in adaptively tolerant T cells.

Figure 8

A, Purified naïve, adaptively tolerant, or pre-activated T cells were stimulated for 2 min with anti-CD3-biotin mAb (0 or 1μg/ml) and anti-CD28-biotin mAb (0 or 5μg/ml) followed by cross-linking with streptavidin. Samples were lysed with 1% NP40 buffer and run on SDS-PAGE. Western blots were performed and probed with an anti-ITK mAb and then reprobed with an anti-actin mAb. The relative values normalized to the total level of actin expression are shown in the right panel.

B, Real-time quantitative RT-PCR analyses of expression of genes encoding Itk, Gads, Rlk and Tec is presented relative to the expression of cyclophilin A in naïve, adaptively tolerant, or pre-activated T cells.

C, Purified naïve, adaptively tolerant, or pre-activated T cells were stimulated for 1 or 5 min with anti-TCRβ-biotin mAb (0 or 1μg/ml) and anti-CD4-biotin mAb (0 or 5μg/ml), followed by cross-linking with streptavidin. Samples were lysed with 1% NP40 buffer and the crude lysate run on SDS-PAGE and immunoblotted with anti-SLP76 Ab, anti-GADS Ab or anti-Grb2 mAb. The lysate was also immunoprecipitated with anti-SLP-76 and run on SDS-PAGE. Western blots were performed and probed with an anti-phosphotyrosine mAb (lowest panel). This experiment was performed 3 times.