Fig. 7. PRL mRNA, PRL, and N-terminal 16K-PRL are detected in chondrocytes and in chondrocyte-conditioned medium. (A) RT-PCR product obtained from rat chondrocytes using primers specific for PRL (lane 3). PRL cDNA was used as a positive control (lane 1), and omission of reverse transcriptase served as a negative control (lane 2). (B) Reducing western blot analysis of immunoreactive PRL-like proteins in chondrocytes. Western blots with a mixture of purified standards (stds) of rat PRL, N-terminal 16K-PRL, and C-terminal 6K-PRL were run in parallel with 200 µg of chondrocyte lysate and were first probed with anti-rat PRL antiserum (anti-PRL), then stripped and reprobed with monoclonal antibody INN-368 directed against the C-terminal end of PRL (C-Term), and subsequently stripped and reprobed with monclonal antibody INN-1 directed against PRL N-terminal region (N-Term). (C) Western blot analysis of chondrocyte-conditioned medium immunoprecipitated with anti-PRL or normal rabbit serum (NRS), and blotted with either anti-PRL or INN-1. Arrows indicate immunoreactive proteins of 17 and 16 kDa. The relative molecular masses of PRL standards are indicated. (D) Proliferation of bovine umbilical vein endothelial cells incubated in the absence (basal) or presence of 10 µl of chondrocyte-conditioned medium alone or together with a 1:400 dilution of anti-PRL or NRS. Values are the mean ± s.e.m. of three independent experiments. ^*P<0.05 vs basal.