Production of leptin receptors (OB-Rb) in normal and OA osteoblasts. The expression of leptin receptors was first determined by qPCR. A) Confluent Ob were lized in TRIzol and RNA extracted as described in Material and Methods. RNA was reversed transcribed followed by PCR amplification of 100 ng cDNA as described in Figure 1 using OB-Rb and GAPDH primers. Results are the mean ± SEM of n = 7 normal and n = 19 OA Ob preparations, P < 0.004 vs normal and OA. B) OA Ob were incubated for 24 hours with increasing concentrations of exogenous leptin. Cells were then lyzed and used for PCR amplification of OB-Rb as in A. Results are the mean ± SEM of n = 6 OA Ob preparations. Second, the production of leptin receptors was determined by Western blot analysis. C) Confluent Ob were treated for 48 hours with or without 1,25(OH)2D3 (50 nM), leptin (100 ng/ml), TGF-β1 (10 ng/ml) or HGF (10 ng/ml). The cells were then lized in RIPA buffer prior to separation using SDS-PAGE and Western blotting using specific antibodies to OB-Rb.