Effect of exogenously expressed cell-surface tmLYVE-1 on 125I-FGF2 binding. (A) Quantitative cell attachment assay of tmLYVE-1 CHO cells. TmLYVE-1 CHO and mock-transfected CHO cells were plated onto microtiter wells coated with HA and cell attachment was analyzed as described in supplemental Methods. The experiment was repeated 2 times in triplicate, values are mean ± SD. (B) 125I-FGF2 binding to low-affinity sites in the tmLYVE-1 CHO clone. TmLYVE-1 CHO and mock-transfected cells were incubated with 10 ng/mL 125I-FGF2 in the presence or absence of 100 ng/mL heparin. After 2 hours of incubation at 4°C, the radioactivity associated with HSPGs was measured as indicated in “Methods.” Data are expressed as a specific activity (125I-FGF2 ng/106 cells). The binding of the figure depicts representative experiment done in duplicates (datapoints as mean ± SD, n = 3). (C) 125I-FGF2 binding to high-affinity sites in tmLYVE-1 CHO-R3. TmLYVE-1 CHO-R3 and mock-transfected cells were incubated with increased concentrations of 125I-FGF2. After 2 hours of incubation at 4°C, the radioactivity associated with FGFR was measured as indicated in “Methods.” Data are expressed as a specific activity (125I-FGF2 ng/106 cells). The binding of the figure depicts representative experiments done in duplicates (datapoints as mean ± SD, n = 3).
