Fig. 3. Promoter analysis of the upstream regions (UTRs) of the mouse 4-1BB. (A) Upstream regions used for report analysis are indicated as PI, PII, and PIII. Translational start site is designated +1. Three distinct 1.8 kbp upstream regions of each exon were cloned into the pGL3-basic vector. (B) EL4 and CTLL-R8 cells were transiently transfected with UTR constructs and pCMVβ-gal as an internal control. PGL3-basic (Basic), promoterless luciferase report, was used as negative control. Cells were treated with anti-CD3 mAb (5 μg/ml), P/I (20 ng/ml and 1 ng/ml), or Con A (10 μg/ml), and luciferase activity was determined after 40 h. Anti-CD3 mAb was coated on culture plates for stimulation. (C) CTLL-R8 cells were transiently transfected with NF-κB mutant(PI-NF-κBmu), AP-1 mutant (PI-AP-1mu), and double mutant (PI-mu) constructs. The transfectants were stimulated with plate-bound anti-CD3 mAb for 40 h. Transfection efficiencies were normalized by β-gal activity and data are representative of three independent experiments. Values are presented as means ± s.e.