Fig. 2. Influence of VEGF on retinal cell proliferation. (A) Effects of VEGF concentrations on BrdU incorporation in vitro. E5-E7 explants were labeled with BrdU for the last 6 hours (n=4 or 5). (B-D) Western blots show sFLK1 (B), VEGF (C) or FLK1-DN (D) expression. Culture media of transfected HEK cells (B,C) or infected DF-1 cell extracts (D) were probed with antibodies against AP (B), VEGF (C) or FLAG tag (D). Controls used were CMV-AP (B,C) or RCAS-AP virus (D). Arrowheads indicate bands with expected molecular weights. (E-G) Immunostaining show effects of AP (E), sFLK1 (F) or VEGF (G) in E5-E7 explants on BrdU labeling for the last 3 hours. Scale bars: 50 µm. (H) Effects of sFLK1- and VEGF-producing cells on BrdU incorporation (3 hours) at E5 in vivo. Black and white bars represent implanted and the contralateral non-implanted eyes, respectively (n=5 or 6; *P<0.03). (I) Effect of FLK1-DN expression on BrdU incorporation in vivo. Retinas infected at HH stage 10 with RCAS-AP (control) or RCAS-FLK1-DN virus were harvested at E5 and labeled with BrdU for 3 hours (n=5; *P<0.04).