Surface expression of CXCR4 on Tesi and Tesi-Tax control cells and binding properties of CXCR4 in the presence of Tax. (A) Tesi and Tesi-Tax control cells were incubated with a polyclonal rabbit anti-CXCR4 antibody followed by a PE-conjugated anti–rabbit secondary antibody. Flow cytometry analyses were performed with a FACScan. (B) Samples containing 5 μg membrane proteins from Tesi or Tesi-Tax control cells and 10 nM of ¹²⁵I-SDF-1 in 100 μL final volume of assay buffer (50 mM HEPES, pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, and 0.5% BSA) were incubated for 90 minutes at 25°C. Bound SDF-1 was separated by filtration through GF/B filters presoaked in 0.5% polyethylenimine. Filters were counted in a Beckman β scintillation counter. Relative ¹²⁵I-SDF-1 binding was calculated by subtracting nonspecific binding measured in the presence of a 100-fold excess of unlabeled SDF-1 from total ¹²⁵I-SDF-1–binding data. Data are reported as the mean ± SD of 2 independent experiments in triplicate.