Fig. 4. The 16K-PRL generated by chondrocytes inhibits endothelial cell proliferation. (A) Coomassie-Blue-stained, non-reducing (NR) SDS-PAGE showing the PRL preparations tested for bioactivity. The preparation containing 16K-PRL was obtained by the incubation of human recombinant PRL with chondrocyte lysates, reduction, and carbamidomethylation as indicated in the methods section. In the preparation with no 16K-PRL, human PRL was added at the end of the incubation of chondrocyte lysates to avoid cleavage, and then the preparation was subjected to reduction and carbamidomethylation. The concentration of PRL in the two preparations was 200 nM and that of 16K-PRL was 100 nM as determined by non-reducing SDS-PAGE densitometry. (B) 16K-PRL inhibition of bFGF-induced proliferation of bovine umbilical vein endothelial cells. Endothelial cells were starved of serum for 24 hours and then allowed to proliferate in complete medium for an additional 24-hour period with or without 2 ng/ml bFGF and the mixture of PRL and chondrocyte proteins that did or did not contain 16K-PRL. Values are the mean ± s.e.m. of triplicate determinations ^*P<0.05 vs bFGF without 16K-PRL. Data are representative of three independent experiments.