FIG 9.

Determination of the functional impact of back substitutions in the background of the front SLAM mutant G71A/N72A. (A) Cell-cell fusion activity in Vero cells triggered by coexpression of CDV H, CDV F and cSLAM (or derivative mutants). To improve the sensitivity of the assay, the cells were additionally transfected with a plasmid encoding the red fluorescent protein. Images of fluorescence emissions from induced cell-cell fusion representative fields are shown. The pictures were taken 24 h posttransfection with a confocal microscope (Fluoroview FV1000; Olympus). (B) Dark-gray bars show the results for cell surface expression of the wt SLAM- and SLAM mutants, determined by treating Vero cells 24 h posttransfection with an anti-HA MAb. After the addition of the secondary antibody, MFI values were recorded by flow cytometry. All values were normalized to the one recorded with the unmodified cSLAM molecule. Means ± SD of data from three independent experiments performed in triplicates are shown. Light-gray bars show the results for quantitative cell-cell fusion assay performed as described in the legend to Fig. 1F. (C) Co-IPs were performed as described in the legend to Fig. 5.