FIGURE 4.

FhitY114 mutants bind poorly to Fhit effector proteins. A, H1299 cells were infected with AdFHIT or AdFHIT mutants at a multiplicity of infection of 5. At 48 h after infection cells were lysed for immunoprecipitation experiments. In the left panel, the immunoblot shows the level of Fhit or mutant Fhit protein expressed in infected cells, normalized to the level of Vinculin. The lysates, in amounts that would give equivalent amounts of Fhit protein, were then used in IP experiments, as shown in the middle and right panels; upper nonspecific bands in these panels include rabbit IgG. Numbers below each lane indicate the relative amounts of Fhit or Fhit mutant protein co-precipitated with Hsp60 or Fdxr, respectively. B, similar experiment was performed using Y114F, Y114A, and Y114D mutants; the left panel shows the input level of Hsp60 and Fhit after infection of H1299 cells. The right panel shows the IP with Hsp60 and Fhit antisera. Western blot images were analyzed with GS800 calibrated densitometer (Bio-Rad) and Quantity One software system.