Figure 3. Synergism between Oct-1 and Prl-R, STAT5 and GR in hormonal induction of β-casein promoter activity.

(A) Diagrammatic representation of the defined transcription factor-binding sites in the LHRR of the rodent β-casein gene. GR, half-palindromic GRE; C/EBP, CCAAT/enhancer-binding protein; TATA, TATA box; YY1, Ying and Yang 1 (a transcription factor). (B) COS-7 cells were transfected with various amounts of mOct-1B/pcDNA3.1 (0–3.0 μg) and expression plasmids of Prl-R, STAT5A and GR together with the LHRR_WT/pGL3, and reporter luciferase activities were determined in extracts from cells stimulated by Prl and Dex for 24 h. (C) In 12-well plates, COS-7 cells were transfected with 0.2 μg of LHRR_WT/pGL3 and 0.2 μg of individual expression plasmids of Prl-R, STAT5A or GR or various combinations of these constructs. The cells were co-transfected with or without mOct-1B/pcDNA3.1, followed by Prl (5 μg/ml) and Dex (0.1 μM) treatment. In all groups, the total amount of DNA was balanced by corresponding vector DNA. Reporter luciferase activities were expressed as the means±S.E.M. for one experiment performed in triplicate. Three independent experiments were carried out. Data from one representative experiment are shown. Bars with different letters are significantly different (P<0.05). The inset in (C) shows the data from Groups 1–6 expanded. (D) Western-blot analysis of whole cell lysates of COS-7 cells transfected with expression plasmids of Oct-1B, STAT5, GR and Prl-R or pcDNA3.1 vector alone and treated with or without Prl and Dex.