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. 2010 Aug 25;24(10):2019–2029. doi: 10.1210/me.2010-0165

Figure 4.

Figure 4

Transcriptional regulation of Smad3 by DHT may occur through mechanisms involving Sp1. A, Deletion map of Smad3-promoter constructs [full-length (FL) promoter = 1892 bp]. NRP-154 or LNCaP cells were transfected with either pGL3-basic [control (cont)] or Smad3 promoter-luciferase (S3p-luc) constructs (FL) or deletions in the presence/or absence of AR (AdMax-AR, 1:500), and incubated with/without DHT for 48 h or the indicated times. Data shown are relative values of firefly luciferase normalized to Renilla luciferase. Each bar represents the average of triplicate determinations ± se. B, Nuclear extracts from samples treated with/without DHT in NRP-154±AR cells were incubated with radiolabeled S3p-Oligo (human Smad3 promoter, nucleotides −612 to −584). For competition assay, nuclear lysates were preincubated for 20 min with either specific competitor (nonlabeled S3p-Oligo) or nonspecific competitors before incubation with the radiolabeled S3p-Oligo. Reactions were then resolved in 6% polyacrylamide-Tris-borate-EDTA gels. C, Separately, nuclear extracts from NPR-154 cells were incubated with biotinylated-S3p-oligo and for DNPAs (Nc, Nuclear extract; Cyt, cytosolic fraction). D, VCaP cells were transfected with Sp1-luciferase and incubated with/without either DHT or R1881 for 48 h. Data shown are relative values of firefly luciferase normalized to Renilla luciferase. Each bar represents the average of triplicate determinations ± se. WT, Wild type.