Identification of a siRNA vector that stably knocks down FKBP12. (A) GFP MFI of 293T cells transfected with eGFP-hFKBP12 (+) or cotransfected with eGFP-hFKBP12 and each of the 17 predicted siRNA sequences targeting the human FKBP12 mRNA or an irrelevant siRNA (irr-siRNA). siRNA4 resulted in > 85% reduction of the GFP MFI. (B) GFP was expressed by 60% of 293T cells transfected with the reporter plasmid eGFP-hFKBP12 alone, by 71% of 293T cells cotransfected with the irr-siRNA plasmid, and by 7% of 293T cells cotransfected with siRNA4 plasmid. (C) The expression of the FKBP12 in nontransduced (NT), irr-siRNA⁺, and siRNA4⁺ EBV-CTLs assessed by WB 1 week after transduction and after selection with puromycin for 3 weeks. FKBP12 expression is visibly reduced in siRNA4⁺ EBV-CTLs after transduction. This effect is more evident after selection in the presence of puromycin. The bottom gel shows the membrane reprobed with anti-GAPDH antibody.