FIG. 2.

Luman induces transcription from the Herp promoter. (A) Schematic structure of full-length and N-terminal Luman as well as the Δ123-186 mutant lacking the basic region responsible for DNA binding. (B) Activation of a Herp promoter (−200/+98) reporter by Luman. 293 cells were transiently transfected with pGL3-Herp-Luciferase reporter together with the reference Renilla luciferase plasmid pRL-SV40 and the effector plasmids encoding Luman(N) and Luman(N)Δ123-186. The vector pcDNA3.1 and treatment with Tm were used as negative and positive controls, respectively. Luciferase values from three independent experiments were normalized to Renilla luciferase activity before being referenced to the control. Cell lysates from the luciferase assays in panel B were subjected to Western blot analysis using Luman antibody M13, shown at the bottom, with β-actin as a loading control. (C and D) Mapping of the Luman-responsive element in the Herp promoter. In both panels C and D, the pGL3-Herp-Luciferase or 10-bp scanning mutation reporter plasmids were cotransfected in 293 cells along with pcDNA3.1 or pcLuman(N). In all scanning mutations of the Herp promoter, nucleotides A, C, G, and T were substituted for C, A, T, and G, respectively. The vector pcDNA3.1 and treatment with Tm were used as negative and positive controls, respectively. The relative luciferase activity was determined by averaging triplicates in three independent experiments, shown with standard errors.