Fig. 5.

T‐Fc is internalized via clathrin‐independent endocytosis. U2OS‐R1 cells were subjected to siRNA‐mediated silencing of clathrin heavy chain (CHC) to inhibit clathrin‐mediated endocytosis. Two different siRNA against CHC were used. Cells were incubated with 100 nm B‐Fc (A) and T‐Fc (B), and fluorescently labeled transferrin as an internal control of CME inhibition for the indicated time periods. Internalized engineered antibodies were visualized with Zenon AF‐488 using confocal microscopy. Scale bars represent 50 µm. (C) Quantification of the effects of CME inhibition on the uptake of B‐Fc, T‐Fc, and transferrin. Internalization of B‐Fc, T‐Fc, and transferrin‐containing vesicles (expressed as integral fluorescence intensity in arbitrary units, AU) was measured with the harmony software. Average values of three independent experiments ±SEM. T‐test was used to assess the statistical significance of measured differences; *P < 0.05, **P < 0.005, n.s.—not significant.