Figure 3

BLU expression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ± SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ± SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit polyclonal antibodies. c-Jun phosphorylation was inhibited by infection with 100 PFU Ad BLU.