Fig. 1

A: Internalization rates of the labeled glyco-pairs of mAb2 into MR-expressing cells. The AF647 labeled symmetrical complex glyco-pair (F1/2, green), the asymmetrical (F3, orange) and symmetrical (F4, blue) high-mannose glyco-pairs of mAb2 were incubated with SUP-B15 cells and SUP-B15 cells without mAbs as control (grey). The AF647 signal of the mAbs due to binding to the cells or internalization into the cells were measured by FACS over time. Data is presented as mean ± SD (n = 8) and fitted with a linear fit with R² > 0.94 to determine the slope. B: Inhibition of the internalization of the symmetrical high-mannose glyco-pair using a competitive cell-based internalization assay. A fixed concentration of the AF647-labeled symmetrical high-mannose glyco-pair (F4) of mAb1 was co-incubated with sequential dilutions of unlabeled mAb3 or unlabeled mannan for 46 h at 37 °C in SUP-B15 cells. After washing the cells, the AF647 signal was measured by FACS. Data is presented as mean ± SD (n = 3) and fitted with a four-parameter non-linear curve. Pointed lines show the upper and lower level of fluorescence. The lower limit is the fluorescence of the cells, and the upper limit is the fluorescence level of the cells with labeled F4 of mAb2, referring to the symmetrical high-mannose glyco-pair. Data is obtained from the mean (n = 6).