FIG. 4.
Detection of myc-U274 on the cell surface by indirect immunofluorescence and FACS analysis. (A) Vero E6 cells transiently transfected with myc-U274 were stained with either the anti-U274 rabbit polyclonal antibody (upper panel) that recognizes the C terminus of the protein or anti-myc monoclonal antibody (lower panel) that recognizes N terminus of the protein. Cells were fixed with 4% paraformaldehyde and used directly (no permeabilization) or permeabilized with digitonin or permeabilized with Triton X-100 before incubation with antibodies. (B) FACS analysis of live Vero E6 cells transfected with myc-U274, myc-U274mut2, myc-GST, or myc-N. Cells were incubated with a myc-monoclonal antibody (unfilled histogram) or an irrelevant antibody of the same isotype (filled histograms), followed by the addition of FITC-conjugated goat anti-mouse F(ab′)2 antibody. Surface expression of myc-U274 caused an increase in surface fluorescence upon staining with myc-monoclonal antibody. The expression levels of the proteins were determined by Western blot analysis (with anti-myc antibody).