Prolactin and Brown Adipocytes Figure 4. IGF-2 is a target gene of PRL in brown adipose tissue. A) qPCR analysis of IGF-2 mRNA expression of BAT from newborn mice, results are expressed as attomol of IGF-2/fmol of 18S, represent mean6SE of 15–16 animals (*** p,0.001). B) Plasma IGF-2 levels. Values in WT (n = 16) and in PRLR KO (n = 21) animals represent the mean6SE. (** p,0.01). C) IGF-2 mRNA is expressed as a function of brown adipocyte differentiation. IGF-2 mRNA expression was determined in immortalized WT, PRLR KO, and PRLR-rescued brown adipocytes at day 0 or day 6 of the differentiation program. Results represent mean6SE of three independent experiments performed in duplicate (*** p,0.001 vs WT at d0). D) Induction by PRL of IGF-2 mRNA expression in T37i cells. Differentiated T37i cells were incubated overnight with 10% horse serum known to lack prolactin and placental lactogens, and were stimulated or not by PRL (100 ng/ml) for 3 h. AG490 (100 mM) was added 15 min prior to PRL exposure. IGF-2 mRNA expression was expressed as mean6SE of 12 independent experiments (* p,0.05). E) STAT5 activation by PRL in T37i cells. Differentiated T37i cells, were stimulated with 100 ng/ml PRL for 5 and 15 min. Protein lysates were subjected to immunoprecipitation with anti-STAT5 antibody. Samples were submitted to a SDS-PAGE and western blotting analysis using anti-phosphotyrosine STAT5 and STAT5 antibodies. F) Specific binding to a STAT response element located in the IGF-2 promoter region. The sequence of STAT-RE is underlined. Nuclear extracts from untreated (8 mg of proteins, lane 1) and PRL-treated T37i cells (8 mg of proteins, lane 2 or 16 mg of proteins, lane 3) were analyzed for binding to a double-stranded STAT-RE oligonucleotide. Specific complexes were identified by competition experiments in which 100 ng unlabeled STAT-RE was added (lane 4). Free probe (lane 5). doi:10.1371/journal.pone.0001535.g004 PLoS ONE | www.plosone.org 7 February 2008 | Issue 2 | e1535