Figure 2

Modified siRNAs allow efficient silencing of ZAP70 silencing mRNA and protein. Stimulated murine T cells were transfected with 0.9 μM ZAP70-specific siRNA pool (Pool) or three different stealth™ siRNAs (S#1, S#2, and S#3) by using nucleofection. Cell lysates were prepared 24 and 48 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control cells were mock transfected ("-", no siRNA added), or transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression in transfected T cells determined 24 h after nucleofection as fold expression of the house keeping gene β-actin. (B) Western blot analysis of transfected T cells 48 h after nucleofection with siRNAs indicated in (A). Protein knockdown of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.