FIG. 5.

The N terminus in conjunction with the CP2b-specific extra 36 amino acids is important for the erythroid cell-specific transcriptional activation of CP2b. (A) The N-terminal region of CP2b is a putative transcriptional activation domain. The yeast one-hybrid assay of the full-length or the N-terminal (aa 1 to 40) deletion form of both CP2a and CP2b fused to LexA BD was performed. The transcriptional activity of each protein was measured by the o-nitrophenyl-d-galactopyranoside assay. (B) The strong erythroid cell-specific transcriptional activity of CP2b requires the broad N-terminal region including the CP2b-specific sequence of 36 amino acids. The mammalian expression plasmids containing the N terminus of CP2 isoforms (aa 1 to 64 or 67), the CP2b-specific sequence (aa 274 to 309), part of the CP2a/b sequence (aa 64 to 274), the N terminus of CP2b spanning the CP2b-specific sequence (aa 1 to 309), the CP2b N terminus plus CP2b-specific sequence (aa 1-64/274-309), or the N terminus of CRTR (aa 1 to 52) fused with the GAL4 DNA binding domain were transiently cotransfected into 293T or K562 cells with a luciferase reporter vector in which GAL4 binding sites are present in the promoter. A dual luciferase assay was employed and firefly luciferase expression was normalized against Renilla luciferase expression. Values are the averages and standard deviations of three independent experiments.