Figure 3.

SOCS2 regulation of GH-induced STAT signalling in osteoblasts and bone. (A) Transcript analysis of Socs1, Socs2 and Socs3 in WT osteoblasts following 24 h GH (500 ng/ml) or IGF1 (50 ng/ml) challenge. Transcript data represented relative to untreated samples as means±s.e.m. Significance denoted by ^c P<0.001. (B) Western blotting analysis of SOCS1, SOCS2 and SOCS3 in WT osteoblasts following 24 and 48 h GH (500 ng/ml) or IGF1 (50 ng/ml) treatment. (C) Western blotting analysis of phosphorylated (P−) STAT1, STAT3, and STAT5 in WT and Socs2 ^−/− osteoblasts challenged with GH (500 ng/ml) for up to 120 min. (D) Immunofluorescence detection of pSTAT5 in WT and Socs2 ^−/− osteoblasts following 20 min GH (500 ng/ml) challenge. (E) Analysis of (P−), STAT5 in SOCS2 overexpressing MC3T3 osteoblast-like cells and negative control-transfected cells following challenge with GH (500 ng/ml) for up to 120 min. (F) Immunofluorescence detection of pSTAT5 in SOCS2 overexpressing MC3T3 cells following 20 min GH (500 ng/ml) challenge. Scale bars for immunofluorescence represent 50 μm. Nucleus, blue; pSTAT5, pink. (G) Analysis of (P−) STAT5 in bone samples extracted from 24-day-old WT and Socs2 ^−/− mice following 15 min GH (3 mg/kg) treatment. Band intensities were quantified by densitometry and analysed for significance, as depicted in the graphs. Data presented as mean±s.e.m. (n=3). Significance denoted by ^b P<0.01.