Figure 4

Conserved residues within the coiled-coil domain of striatin are critical for association with PP2A but not with Mob3. (A) HEK293 cells transfected with HA-tagged wild-type and coiled-coil mutant striatins were lysed and HA-striatin immune complexes were isolated and proteins contained in the immunoprecipitates were detected by immunoblotting using antibodies that recognize the HA-epitope tag, PP2A C subunit, Mob3, and SG2NA. (B) Relative binding of wild-type and point mutant striatins to PP2A C subunit, Mob3, and SG2NA was measured as described in the legend to Figure 2B. The error bars represent the standard deviation of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01 relative to wild-type. (C) A similar experiment to (A) was conducted with wild-type striatin and striatin mutants with hydrophobic coiled-coil residue substitutions. (D) Relative binding of wild-type and point mutant striatins to PP2A C subunit, Mob3, and SG2NA was measured as described in the legend to Figure 2B. The error bars represent the standard deviation of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01 relative to wild-type. The difference in C subunit association between L84A/L94A/I102A and L84A/L94A/L105A was also significant (p = 0.029). (E) A similar experiment to (A) was conducted with wild-type striatin and small, coiled-coil-containing mutants, P132Stop striatin and 46-131 striatin. The HA-striatins migrated at different positions but are shown side by side for comparison of their levels. Although some non-specific sticking of C subunit was seen in the vector control lane, both mutants bound wild-type levels of PP2A C subunit.