Stat5-dependent β-casein promoter activity is enhanced in the presence of TSA.

(A) 293T cells were transfected in triplicates with 0.6 μg reporter plasmid (β-casein promoter luciferase) together with the expression plasmids for mStat5a, (pXM-Stat5a; 0.6 μg), and prolactin receptor (pcDNA3-prolactin receptor; 0.3 μg) using a calcium phosphate method. Cells were stimulated with or without 5 μg/mL prolactin and 200 nM TSA and equal amounts of the cell lysates were assayed for luciferase activity. Luciferase activity assayed in cells treated with prolactin was given as fold induction over values obtained without prolactin, which were set as 1. Transcriptional induction is given as the mean and SD of 5 experiments. The corepressor SMRT binds to wild-type Stat5a. (B) Cellular extracts from 293T cells transfected with 5 μg HA-tagged Stat5a expression plasmid (pXM-HA-Stat5a) were incubated with radiolabeled SMRT. Subsequent immunoprecipitations were performed with anti-HA monoclonal antibody (α-HA), anti-FLAG antibody (α-Flag), or protein A/G Sepharose alone (A/G Seph). Radiolabeled SMRT protein was detected on a dried gel in extracts precipitated with α-HA antibody, whereas Stat5a was detected by Western blot using anti-HA antibody and both were compared to 10% input.