Figure 3.

Binding affinity and internalization of GCSFR isoforms. (a) Ba/F3 cells expressing class I or class IV cells were equilibrated with increasing concentrations of ¹²⁵I-labeled hGCSF for 1 h at RT. Bound *GCSF was separated from the free. Data were plotted as bound vs free and the binding affinity was determined using nonlinear curve fitting based on a one site-specific binding model (Table 2). (b) Receptor internalization of class I, class IV and d715 GCSFR was determined by pre-equilibration with a mouse IgG1 anti-human CD114 Ab for 1 h at 4 °C followed by incubation with hGCSF at 37 °C for 0, 7.5, 15, 30 and 60 min. Ba/F3 parent cells were used to determine the background for each time point. Internalization at various times was determined by quantitating the number of surface receptors by flow cytometry, using a PE-conjugated rat anti-mouse IgG1 secondary antibody. The data are plotted as background subtracted geometric mean fluorescence instensity normalized to time 0, set at 100%, against time. Nonlinear curve fitting using a single phase decay was performed using GraphPad Prism v5.04 software. Statistical analysis was performed applying Bonferroni’s multiple comparison test after two-way analysis of variance. Statistically significant differences between class I and class IV at 7.5 and 15 min of internalization were observed and represented as *P<0.05 and **P<0.01, respectively. PE, phycoerythrin