Figure 3.

Chordin-like 1 expressed in human retinal pericytes is secreted and binds to bone morphogenetic protein-4. A: Conditioned media from HRPC exposed to 1% O₂ for 24 and 48 h was examined by western blot analysis for secretion of CHL-1, using an anti-CHL-1 antibody. B and C: BMP-2 and BMP-4 expression in HRPC was examined in cells cultured in normoxia (N) and hypoxia (H) by RT–PCR (B) and secreted BMP2 and BMP-4 were detected in conditioned media from HRPC exposed to 1% O₂ for 24 and 48 h (C). D: Transfection of the expression vector pcDNA6/CHL-1 V5-His into Cos7 cells, using the transfection reagent Fugene 6, resulted in expression of an approximately 60 kDa protein, which was detectable using an anti-V5 antibody. Cells were transfected with either an empty vector (E), pcDNA6/V5-His C, or a V5 tagged CHL-1 expressing vector (CHL-1), pcDNA6/CHL-1 V5-His. E: Whole cell extracts from Cos7 cells were transfected, using the transfection reagent Fugene 6, with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 and 100 ml NiNTA magnetic beads at 4 °C overnight. The complexes were washed, the beads and examined by western blotting for the presence of CHL-1, using anti-V5 antibody, and BMP-4, using an anti-BMP-4 antibody.