Figure 2

The N-terminal region of striatin binds PP2A while Mob3 associates with both N-terminal and more C-terminal striatin sequences. (A) HEK293 cells transfected with HA-tagged wild-type and mutant striatins were lysed and HA-striatin immune complexes were isolated, separated by SDS-PAGE, and proteins contained in the immunoprecipitates were detected by immunoblotting using antibodies that recognize the HA-epitope tag, PP2A C subunit, and Mob3. (B) Relative binding of PP2A C subunit and Mob3 to wild-type striatin and C-terminal truncation mutants of striatin was determined by quantitatively comparing the C subunit/HA-striatin and Mob3/HA-striatin ratios of wild-type striatin to those of the mutants using a Bio-Rad Fluor S-Max chemilumimager (see Methods). (C) Immunoblot showing that Mob3 also associates with residues 310-780 of striatin while PP2A does not. (D) Relative binding of PP2A C subunit and Mob3 to wild-type and Δ(3-309) striatins was measured in the same manner as described in B. The HA-striatins migrated at different positions due to their size differences but are shown side by side in panels A and C for comparison of their levels. C subunit can migrate as either singlets or doublets; whether double or single bands are seen can vary for the same sample from gel to gel. This pattern of migration in SDS-PAGE has been noted previously for endogenous PP2A C subunits [40–43] and is not due to degradation. The error bars in panels B and D represent the standard deviation of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01 relative to wild-type.