Fig. 2. Binding of YFP-IL-6 to gp130 deletion mutants. (A) COS-7 cells were transfected with an expression vector encoding human gp130/id-CFP, gp130/D1id-CFP or gp130/D2,3id-CFP (lower schemes). The cytoplasmic parts of the gp130 constructs are deleted after Pro668. Therefore, the constructs lack the dileucine internalization motif resulting in enhanced surface expression (id, internalization deficient). Cells were fixed 48 hours after transfection. Non-permeabilized cells were stained for immunofluorescence using the antibody B-T2 against D1 (upper row), the antibody B-P8 against CBM/D2,D3 (middle row) or the antibody B-P4 against D4 of gp130 (lower row) followed by a PE-coupled secondary antibody. In the confocal images PE fluorescence is red and CFP fluorescence is blue. COS-7 cells were transfected with an expression vector encoding gp130/id-CFP (B), gp130/D1id-CFP (C) or gp130/D2,3id-CFP (D). 48 hours after transfection, living cells were incubated in a perfusion chamber at 37°C. Confocal images were taken 10 minutes after addition of YFP-IL-6 (40 ng/ml) and sIL-6R (1 µg/ml). Bar, 20 µm.