Figure 2
From: Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1
Binding of Caveolin-1 Peptides to NSP4 Constructs. Panel A: Transduced yeast lysates expressing full-length- NSP4,-NSP4-Ala6 and -NSP4-HydroMut (FLNSP4, FLNSP4-Ala6, and FLNSP4-HydroMut) were incubated with CNBr-activated sepharose 4B beads bound by the N-terminal (cav-12-31), C-terminal (cav-1161-178), or beads only. The beads were washed and the captured proteins separated by SDS-PAGE, transferred to nitrocellulose, and probed using rabbit anti-NSP4150-175. Controls (lanes 1-4) show the absence of non-specific binding to the sepharose beads with all lysates tested. Lanes 5-7 show the reactivity of FLNSP4 and-NSP4 mutants with cav-12-31. Only the FLNSP4-HydroMut failed to bind the N-terminal caveolin-1 peptide. The same binding pattern was observed when FLNSP4 and -NSP4 mutant proteins where incubated with cav-1161-178 (lanes 9, 10, and 11). InVSc1 alone showed no NSP4-specific bands using either peptide (lanes 8 and 12). Panel B: Western blot analyses of CNBr-activated sepharose 4B beads that were bound by cav-12-31, cav161-178 or no peptide and reacted with yeast lysates expressing Rev113I, Rev124V, Rev131Y. The peptide-bound proteins were detected by Western blot using rabbit anti-NSP4150-175. Lanes 1-4 demonstrate that the NSP4 proteins failed to bind the sepharose beads alone with all lysates tested. Lanes 5, 6, and 7 indicate that all three revertant NSP4 proteins bound to the N-terminal peptide, Cav-12-31. The same binding pattern was observed with the C-terminal peptide, cav-1161-178 (lanes 9, 10, and 11), while InVSc1 failed to bind either peptide (lanes 8 and 12).