FIG. 5.

Binding of Gs lacking the HBD to HEp-2 cells. Partial sequences of the Gs wild-type and GsΔHBD proteins are shown at the top, denoting the proposed HBD (framed) and the amino acids deleted in the latter protein. (A) Western blot analysis (using antibody 63G) of 20 μl of wild-type Gs and mutant GsΔHBD that were concentrated from the supernatants of HEp-2 cells infected with vaccinia virus recombinants expressing the corresponding proteins. (B) The indicated amounts of each protein were assessed for their ability to bind to HEp-2 cells by flow cytometry (shaded histograms). The unshaded histogram is a negative control representing cells incubated without Gs. (C) HEp-2 cells were either incubated with 80 mIU of heparinase I/ml or mock digested as indicated in Fig. 4 before being incubated with 30 μl of Gs (black) or GsΔHBD (grey) and subjected to flow cytometry to assess attachment. The results are expressed as the percentage of mean fluorescence obtained relative to that produced by the binding of Gs to untreated HEp-2 cells after subtraction of background values (without added Gs).