Figure 3.

Belimumab binds to membrane-bound BAFF in furin-deficient U937 cells. (A) Wild-type (WT), furin-deficient (furin-ko) and BAFF-deficient (BAFF-ko) U937 cells were stained with the indicated amounts of biotinylated atacicept after saturation of Fc receptors with human IgGs. Mean fluorescence intensity (MFI) of staining was monitored. Single measures were performed. Experiment performed four times. (B) Same as panel A, except that staining was performed with biotinylated belimumab. Experiment performed four times. (C) The indicated U937 cells were washed and then put in culture. Human BAFF was quantified by ELISA in conditioned cell supernatants collected at the indicated time points. LLQ: lower limit of quantification. Double-sided arrows indicate the fold difference between concentrations of BAFF in supernatants of wild type and furin-deficient U937 cells. Error bars of measures performed in duplicate (mean ± SEM) are smaller than symbol size. Experiment performed twice. (D) BAFFR:Fas reporter cells were exposed to titrated amounts of the same conditioned supernatants used in panel C. After an overnight incubation, cell viability was monitored with the PMS/MTS assay. Single measures were performed. Double sided arrows indicate the fold difference between EC₅₀ of wild type and furin-deficient supernatants. One measure indicated in brackets was excluded for the determination of EC₅₀. Experiment performed twice in this format, and two more times with a single time point.