FIGURE 3.

The N-terminal regulatory determinant is transferable. A, attaching the first 41 amino acids of TPH2 to the N terminus of HA-TPH1 results in a chimeric protein with expression levels most similar to those of wild-type HA-TPH2 in both HEK293 and PC12 cells. Because the vector used to make the constructs contains a neomycin resistance gene (neomycin phosphotransferase), a blot for this protein was used as a transfection control in HEK293 cells. The graph shown in B is the result of a densitometric analysis of blots from HEK293 cells (n = 3) with results presented in arbitrary units normalized to neomycin phosphotransferase levels. ^*, p < 0.01 compared with HA-TPH1; ^**, p < 0.05 compared with HA-TPH2. C and D, insertion of the first 35, 41, or 58 amino acids of TPH2 after the HA epitope of an HA-GFP construct results in decreased HA-GFP expression levels in PC12 cells. Unlabeled GFP was included to assess antibody specificity. Results presented are normalized to HA-GFP levels (n = 4). ^***, p < 0.001 compared with HA-GFP. E, there is no difference in mRNA levels between the HA-GFP construct and the representative chimera, HA-(1–35)-GFP, as measured by quantitative reverse transcription-PCR (n = 4). All graphical data represent means ± S.E.