formation. Moreover RT-PCR products were analyzed in a post- amplification fusion curve to ensure that a single amplicon was obtained. Quantification was performed by the standard curve method. Standard curves were generated by serial dilutions of a linearized plasmid containing the specific amplicon, spanning six orders of magnitude, yielding a correlation coefficient of at least 0.98 in all experiments. For all experiments, PCR efficiency was close to 1, indicating a doubling of DNA at each PCR cycle, as expected. Ribosomal 18S RNA was used to normalize for RNA quality, quantity and RT-efficiency. Relative expression within a given sample was calculated as the ratio (attomol of specific gene/fmol of 18S). Results are expressed as mean6SE and represent the relative expression compared to that obtained with control animals or cells. Kaplan-Meier All experiments and animal care were performed in accordance with the animal facility of the Faculty of Medicine Necker. Cold tolerance was accessed by housing newborn mice at 4uC. When no sign of tactile stimulation was observed, the mice were sacrificed. We found that mice up to 6 h were considered without using death as an end-point. The survival rate was calculated and expressed according to Kaplan-Meier representation. Western blot analyses Total protein extracts were prepared from BAT isolated from WT and PRLR KO mice. Tissues were washed twice with ice-cold PBS and lysed as described in [18], then 2 mg of total protein were directly submitted to SDS-PAGE. After protein blotting on a nitrocellulose membrane (Biorad, Marnes-La Coquette, France), blots were incubated for 1 h at room temperature in 5% milk-Tris buffer saline/ 0.1% Tween before incubation with the goat anti- UCP1 (1:3000) (a gift from Pr. D. Ricquier) overnight at 4uC. After extensive washes, membranes were incubated with a goat peroxidase-conjugated second antibody (1:2000) for 1 h at room temperature and proteins were visualized as mentioned above. After milk saturation, blots were reincubated overnight with rabbit anti-ERK1/2 (1:1000) (Upstate Biotechnology, Charlottesville, VA) then incubated with a rabbit peroxidase-conjugated second antibody (1:10000) for 1 h at room temperature and proteins were visualized by use of the ECL+ detection kit. IGF-2 measurement IGF-2 was measured by RIA [46] after extraction of IGF binding proteins on a BioGel P-10 (Bio-Rad, Hercules, CA) column equilibrated in 1 M acetic acid. The method has been validated for complete separation of IGFBPs from IGFs in serum and other biological fluids. The IGF-2 assay uses recombinant human IGF-2 as standard and tracer and a mouse monoclonal antibody. The intra- and inter-assay coefficients of variation are less than 10%. The sensitivity of the IGF-2 assay is 50 pg/ml. Immortalization of WT and PRLR KO preadipocytes Brown adipocytes and their precursor cells were isolated from newborn mice by collagenase (1 mg/ml) digestion (Serva, Heidel- berg, Germany) for 15 min at 37uC as described in [47]. Preadipocytes were thereafter immortalized by stable transfection with 2.5 mg of HA-TAg SV40 plasmid and 0.5 mg of pEGFP plasmid (Clontech, Palo Alto, CA) using Lipofectamin Reagent Plus (InVitrogen). The plasmid HA-TAg SV40, encoded the Large T Antigen of the simian virus 40 placed under the control of the proximal promoter of the human mineralocorticoid receptor gene. This plasmid was originally used as a transgene and allowed to Prolactin and Brown Adipocytes generate the T37i cell line derived from a hibernoma of a transgenic mouse [20]. The pEGFP plasmid encoded for the enhanced green fluorescent protein and for the NeoR gene. Transfected cells were selected in the presence of 400 mg/ml of geneticin (G418, InVitrogen). Selective medium was changed 3 times a week for 2 weeks. Foci of cells were isolated using cloning rings and expanded when possible. PRLR-rescued brown adipocytes were obtained by transfecting PRLR KO cells with a 2.2 kb mouse PRLR cDNA previously subcloned into pcDNA 3.1 hygro expression vector (InVitrogen) after selection with 200 mg/ml hygromycin. Cell culture All established cell lines but also T37i brown adipose cell line were cultured in a complete medium composed of DMEM/HAM’s F12, 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 20 mM HEPES and were grown at 37uC in a humidified atmosphere with 5% CO2. Differentiation into mature brown adipocytes was achieved by incubating subconfluent cells with complete medium supplemented with 2 nM triiodothyronine (Sigma, Saint Quentin Fallavier, France) and 20 nM insulin for 7 days. All products were purchased from InVitrogen except when stated. Before each hormonal treatment, human PRL was produced by recombinant technology then purified as described previously [48]. Human IGF-2 was purchased from Calbiochem (La Jolla, CA). Immunodetection of the SV40 large T antigen Immunodetection was performed using a monoclonal anti-SV40 large T Antigen antibody (Ab-2; Calbiochem). Cells were seeded on glass slides and fixed with 100% ethanol, rinsed with PBS, and incubated with a 1:100 dilution of the primary antibody for 45 min at 37uC. After washing, cells were incubated for 30 min at 37uC with a 1:150 dilution of the biotinylated goat anti-mouse antibody (Tebu, Le Perray en Yvelines, France) followed with an incubation for 30 min at 37uC with a 1:100 dilution of CY3-streptavidine (Jackson ImmunoResearch Laboratories, West Grove, PA). Studies of the activation of STAT5 signaling pathway Differentiated T37i cells, starved for 18 h in minimum medium, were stimulated with 100 ng/ml PRL, during 0, 5 and 15 min; cells were washed twice with ice-cold PBS and lysed as previously described [18]. Five hundred mg of total protein were immuno- precipitated overnight at 4uC with protein A sepharose (Amer- sham) and the rabbit polyclonal anti-STAT5 (C17 Santa Cruz), reduced in Laemmli buffer and a 8% acrylamide SDS-PAGE was performed. Electrotransferred proteins were incubated with monoclonal anti-phosphotyrosine (1/5000 clone 4G10 Upstate Biotechnology) followed by incubation with the secondary antibody: anti-mouse IgG peroxydase-linked (GE Healthcare), detected with ECL western blotting detection reagent (GE Healthcare) and exposed to a film (Konica X-ray film AX, Roissy, France). Blots were stripped by a 30 min-incubation at 50uC in 62.5 mM Tris-HCl pH 6.8, 2% SDS, 100 mM 2-mercaptoetha- nol, washed with TBS/0.1% Tween 20 and were incubated with anti-STAT5 and anti-rabbit IgG peroxydase-linked (GE Health- care) as secondary antibody. EMSA Cells were washed with cold PBS and homogenized in 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 600 mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol and 0.1% PLoS ONE | www.plosone.org 11 February 2008 | Issue 2 | e1535