Fig. 3.

BFA inhibits the interaction of CK1ɛ - Dvl2.

(A) BFA reduces the phosphorylation of Dvl2 and the amount of Plk1 during primary cilium disassembly. hTERT-RPE cells were treated with either the DMSO control or BFA as described in Fig. 2. Cells were harvested and subjected to immunoblotting analysis with the indicated antibodies. α-tubulin was used as a loading control. (B) BFA does not inhibit the activity of CK1ɛ. HEK293T cells transfected with Myc-tagged CK1ɛ were harvested at 24 h after transfection and subjected to immunoprecipitation with anti-Myc antibody. Precipitates were subjected to in-vitro kinase assay in the presence of γ-³²P ATP. The DMSO control, BFA, or D4476 was treated to in-vitro kinase assay mixture. (C) BFA inhibits the binding of CK1ɛ to Dvl2. Bacterially purified GST-CK1ɛ proteins were incubated with Flag-Dvl2-expressing HEK293T cell lysates in the presence of the DMSO control or BFA and GST pull-down assay was performed. (D) BFA interferes with the binding between CK1ɛ and Dvl2 in in-vivo conditions. HEK293T cells co-transfected with Myc-tagged CK1ɛ and Flag-tagged Dvl2 were harvested at 48 h after transfection and subjected to immunoprecipitation with anti-Myc antibody. Precipitates were subjected to immunoblotting analysis with the indicated antibodies. BFA was applied to cells either 6 h or 24 h prior to cell harvest. Note that exogenous Dvl2 does not make a double band, only endogenous Dvl2 makes a double band.