Stimulation is required to silence ZAP70 expression in T cells. Murine T cells were stimulated ("+", anti-mouse CD3ε) or left untreated ("-"). 24 h later, cells were transfected with 0.9 μM ZAP70-specific siRNA pool by nucleofection. Cell lysates were prepared 24, 48 and 72 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control, cells were mock ("-", no siRNA added) transfected ( ) or cells were transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression determined as fold expression of the house keeping gene β-actin. (B) Western blot analysis 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 four independent experiments.
Gust et al. Cell Communication and Signaling 2008 6:3 doi:10.1186/1478-811X-6-3