Figure 3

Nck1 is required for TCR-induced CD69 expression in primary cells. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat derived from healthy blood donors. Cells were treated with siRNA specific to either Nck1 or Nck2 and treated with both siRNA specific to Nck1 and Nck2. A) The expression of Nck was analysed by immunoblotting. B) The expression levels of surface CD3 molecules on these knockdown primary cells were measured by flow cytometry. C-D) siRNA-treated cells were stimulated either with anti-CD3 antibody or PHA/PMA for 24 hr. Cells were then harvested and stained with PerCP-conjugated mouse anti-human CD4 mAb and PE-conjugated mouse anti-human CD69 antibody before analysing by flow cytometry. Numbers in CD69 histogram indicate frequency of positive cells within a CD4 gated population. Grey shaded histrogram and grey letter are cells treated with control siRNA, black bold solid line and black letter are cells treated either with siRNA specific to Nck1 or Nck2, and black dotted line is isotype control staining.