FIGURE 3.
The hSloΔ579–664 variant is a dominant negative of cell surface expression. a, Western blot of total cell lysates from the FLAG-hSloΔ579–664-HA and FLAG-zero-HA variants expressed in HEK293 cells. b, representative blots from co-immunoprecipitation (IP) experiments from HEK293 cells co-expressing the zero-HA variant with either the FLAG-hSloΔ579–664 or FLAG-e22 variants. Left panel, channels were immunoprecipitated with mouse anti-FLAG M2 antibody, and blots were probed with rabbit anti-HA antibody. Right panel, channels were immunoprecipitated with rabbit anti-HA antibody and probed with mouse anti-FLAG M2 antibody. c, zero-HA subunits were co-expressed with either FLAG-hSloΔ579–664-eYFP, FLAG-e22-eYFP, or FLAG-zero-eYFP channels in HEK293 cells, and cell surface (FLAG) expression was quantified. d, FLAG-zero-eYFP subunits were co-expressed with either hSloΔ579–664-HA, e22-HA or zero-HA channels in HEK293 cells. In c and d, the data are the cell surface FLAG staining values expressed as percentages of control (surface FLAG-zero-eYFP levels when co-expressed with the zero-HA construct) under nonpermeabilized conditions. e, representative Western blots of HA or green fluorescent protein immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the indicated constructs and corresponding whole cell lysates. All data are the means ± S.E. from a minimum of six independent experiments with >700 cells analyzed/group in c and d and three independent experiments in a, b, and e. **, p < 0.01, ANOVA with post hoc Dunnett's test compared with other groups.