Figure 3.

Freud-1 represses basal 5-HT1A receptor expression in raphe RN46A cells via FRE. Luciferase activity was normalized to that of β-galactosidase and is expressed as relative light units and normalized to control. *p < 0.05 compared with control by t test. A, CalB-dependent Freud-1 repression. The FRE-containing -2300 5-HT1A-luciferase was transiently cotransfected in L6 myoblast or raphe RN46A cells with vector (pcDNA3, Control), Freud-1, or Freud-1 del mutant of the C2 (CalB) domain. Above, A Western blot of 30 μg of nuclear extracts was probed with anti-Flag to show equal expression of Flag-Freud-1 or Flag-Freud-1 del in these experiments. B, FRE dependence of Freud-1 repression. Transfections as above were done with FRE-inactivated mutant 2300m1, which was insensitive to Freud-1. C, FRE-dependent repression of SV40 promoter by Freud-1. The 31 bp DRE (DRE/SV40) or the FRE-mutant DRE (DREmut/SV40) was placed upstream of the SV40 promoter and cotransfected with vector (pcDNA3) or Freud-1 expression construct. D, E, Freud-1 inhibits transcriptional activity of the 5-HT1A receptor gene. L6 myoblast or raphe RN46A cells were transiently cotransfected with the DRE-containing (-1590luc) 5-HT1A-luciferase reporter or vector (pcDNA3, Control), and sense (D) or antisense (E) Freud-1 expression vectors. Freud-1 protein expression in each cell line after transfection was detected by Western blot using specific anti-Freud antibody (shown above). β-Actin immunoreactivity was tested to confirm equal loading. F, G, Freud-1 protein inhibits 5-HT1A receptor expression in raphe RN46A cells. F, Proteins prepared from RN46A cells transfected with Freud-1 sense or antisense expression vectors and differentiated for 72 hr were subjected to anti-5-HT1A receptor immunoblotting. β-Actin immunoreactivity was tested to confirm equal loading. Shown is a representative blot of three independent experiments. The relative intensity was quantified by an Automated Digitizing System (UN-SCAN-IT, Silk Scientific Inc.). Data represent the mean ± SE of three independent experiments. G, RN46A cells cotransfected with 1 μg of Red Fluorescent Protein (DsRed) and 5 μg of Freud-1 sense or antisense expression plasmids as indicated were stained for 5-HT1A immunoreactivity (see Materials and Methods). Arrows indicate representative DsRed-positive cells that express either high or low 5-HT1A immunoreactivity (for antisense or sense Freud-1, respectively) compared with cells not transfected with DsRed.