Western blotting studies were performed essentially as previously published.31,33 Y79 cells were pelleted by centrifugation (900 rpm for 5 minutes) and suspended in lysis buffer (100mM Tris, 150mM sodium chloride, 1mM EDTA, 1mM sodium vanadate, 1mM sodium fluoride, 0.2M phenylmethanesulfonyl fluoride, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, and protease inhibitor cocktail, 100 μL/sample), to which was added an equal volume of Laemmli buffer. A human breast cancer cell line (SUM44PE, generous gift of S. Ethier, PhD, University of Michigan Breast Cell/Tissue Bank and Database, Ann Arbor) that overexpresses FGFR1 was also collected as a positive control.40 In addition, human retinal samples (postmortem retina within 24 hours of death) were solubilized in lysis buffer as described. Equal amounts (50 μg per well) of solubilized proteins were loaded onto polyacrylamide gels and separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membranes and blocked with TRIS-buffered saline (TBS), 5% dry milk, 3% bovine serum albumin, and 0.2% Tween 20 for 1 hour at room temperature, then incubated with the 4 FGFR polyclonal antisera listed in the preceding section, each diluted 1:2000 in TBS and 0.2% Tween 20 overnight at 4°C. Bound primary antibody was detected by means of peroxidase-conjugated goat anti–mouse secondary antibody (Jackson Immunoresearch Laboratories, West Grove, Pa), 1:15 000 dilution in TBS and 0.2% Tween 20. Immunoreactive bands were visualized with a substrate kit (Pierce Super Signal Substrate Kit; Pierce Biotechnology, Rockford, Ill) according to the manufacturer’s instructions.