Figure 3
From: Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1
Representative Western blot analyses of yeast lysates co-transformed with NSP4 or NSP4 mutants with caveolin-1 fusion proteins. The presence of NSP4- and cav-1 fusion proteins in the Y2H assay was shown by Western blot analyses of the yeast co-transformed with pD22cav-1 and each of the following fusion proteins: pD32NSP4 46-175 (lanes 2A and 2B), pD32NSP4-Ala6 (lanes 3A and 3B) , pD32NSP4-HydroMut (lane 4), pD32Rev113I (lane 5), pD32Rev124V (lane 6), and pD32Rev131Y (lane 7). Lanes 8 and 9 show the FLNSP4-HydroMut and FLNSP4, respectively. Blots were probed using (A) rabbit anti-NSP4150-175 or (B) rabbit anti-cav-12-31. Panel A: The non-transformed yeast (MaV203, lane 1) showed a non-specific band at ~29 kD that was observed in all lanes. Lysates expressing FLNSP4-HydroMut (lane 8) demonstrated NSP4-specific bands at ~ 28, 24 kD. Lysates expressing FLNSP4 showed bands at ~ 28, 24, and 20 kD (lane 9). All of the co-transformed yeast demonstrated a NSP4-fusion protein band at ~ 34.4 kD (A, lanes 2-7). Panel B revealed a faint contaminating band below the 34.9 kD specific caveolin-1-fusion protein band (lanes 1-7). Lanes 2-7 show the specific caveolin-1 fusion protein band at ~34.9 kD. Panel C: Lysates expressing FLNSP4 (lane 1) showed bands at ~ 24 and 28 kD and (lane 9). All of the co-transformed yeast demonstrated both monomeric and multimeric NSP4-specific fusion protein bands (lanes 1). Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed the ~34.9 kD specific NSP4-1-fusion protein band (lanes 2-5). Also, Rev1M124,127A demonstrated a multimeric NSP4-fusion specific band similar to one observed with FLNSP4. Panel D: Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed a ~ 34.9 kD specific caveolin-1-fusion protein band (lanes 1-4). Also, Rev1M124,127A demonstrated a multimeric caveolin-1-fusion protein band.