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.