(A) Syn5-Bet1 interaction is enhanced upon K270R mutation. HeLa cells expressing indicated constructs were synchronized in mitosis and immunoprecipitated with an anti-Myc antibody and blotted for indicated proteins.
(B) Quantitation of Bet1 or GS28 interaction with Syn5-Myc in (A) from three independent experiments.
(C–E) Syn5 ubiquitination inhibits Syn5-Bet1 interaction in vitro. MBP-Syn5 was pulled down after in vitro ubiquitination reaction and further incubated with recombinant Bet1 (C). After re-isolation of Syn5, bound Bet1 was assessed by Western blot (D). (E) Quantitation of (D) for Bet1 binding to Syn5 from three independent experiments. Left panel: lane 2 vs. 3 in (D). Right panel: lane 3 vs. 4 in (D).
Data are represented as mean ± s.e.m. *, p < 0.05; **, p < 0.01; ***, p <0.001.