FIG 6.

SAM polymerization is required for Bicc1 accumulation and silencing activity. (A) Time course analysis of HA-Bicc1 WT and mutants expressed in HEK293T cells after CHX treatment. HA-Bicc1 protein levels were compared with γ-tubulin levels by Western blotting at 0, 8, 24, 32, 48, and 56 h. The relative level of each protein is presented in the graph at the bottom, and the estimated half-life is given on the right. AU, arbitrary units. (B) Level of the HA-Bicc1 WT and mutants upon transfection with a single dose (×1) or a double dose (×2) of DNA encoding HA-Bicc1. A double dose of transfected DNA encoding HA-Bicc1 mutD, the ΔSAM mutant, or the bpk mutant is required to obtain a protein level comparable to that of the HA-Bicc1 WT. The relative percentage of WT Bicc1 is indicated for each condition. γ-Tubulin was used for normalization. (C and D) Silencing of AC6 and PKIα 3′ UTR luciferase reporters by WT or polymerization mutant Bicc1 in HEK293T cells. β-Galactosidase was used as a control for normalization. Error bars show SEMs. *, P < 0.005. (E) Induction of the TOPflash reporter of Wnt signaling by Dishevelled 2 (Dvl2) in transfected HEK293T cells is inhibited by both WT and polymerization mutant Bicc1. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.