a. CD5 promoter deletion constructs. The 3 kb CD5 5-flanking region fragment was excised from p12-1 and subject to 5' to 3' deletion with SspI (-785), RsaI (-530), AciI (-257), StuI (-215), HincII (-172), HinfI (-67). The deleted CD5 5'-flanking regions were cloned into a promoterless luciferase construct, pGLB (Promega). The DNA base pair on the CD5 promoter, before the translation start, is labeled as -1.
b. A 43 bp region in the CD5 promoter is responsible for normal expression in EL4 (T) cells. EL4 cells were transiently co-transfected with each deletion construct and pON405, a LacZ reporter construct, as described in Materials and Methods. Luciferase activity in cell extracts was measured between 36 and 48 hours after transfection. The measurement for luciferase activity was normalized based on the β-galactosidase activity in each cell extract. Each bar value represents the average of three independent transfections in one experiment, with standard deviation as error bars. Each experiment was repeated several times.