FIGURE 3.

Localization and co-immunoprecipitation of mutant Fhit proteins in stable expressors. A, mitochondrial and cytosol fractions of MKN74 transfectants were isolated using mitochondria/cytosol fractionation kit (BioVision), and fractionated lysates were separated on a polyacrylamide gel and probed with anti-Fhit serum (left panel). Western blot images were analyzed with GS800 calibrated densitometer (Bio-Rad) and Quantity One software system to determine the ratio of Fhit or mutant Fhit protein in mitochondria versus cytosol (right panel). Note that the Y114D residue adds a negative charge to the protein, effecting slower migration on the gel, whereas the Y114F and Y114A mutants migrate faster than WT Fhit. Note also that the Y114D mutant was made to mimic a phospho-Fhit and is apparently much less stable than WT, in accord with the report that phospho-Fhit is targeted for degradation (15). B, MKN74/FHIT and FHIT mutant transfectants (WT, Y114A, Y114D, Y114F, H96N, and L25W) were cultured, and 10⁶ cells of each were collected. Cells were lysed; DSP cross-linker was added, and immunoprecipitation with Fdxr or Hsp60 antiserum was carried out; proteins were separated on polyacrylamide gel and probed with anti-Fhit serum. Western blot images were analyzed with GS800 calibrated densitometer (Bio-Rad) and Quantity One software system.