XB-IMG-139105
Xenbase Image ID: 139105
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Fig. S2. Custos is a binding partner of Dvl. (A) Custos binds to Dvl2. See Fig. S3 for schematic diagrams of the Custos and Dvl2 proteins and mutants used. HACustos
and Flag-Dvl2 were transfected into HEK-293T cells and the proteins were immunoprecipitated from lysates and resolved by SDS/PAGE and Western
blotting using the indicated antibodies. Coexpression of Custos with Dvl induced a band-shift of Custos, which is an unphosphorylated form of the protein. (B)
Custos-Dvl complex formation was decreased with Wnt stimulation. HA-Custos was transfected into HEK-293T cells and cells were treated with 250 ng/mL
recombinant mouse Wnt3a. Endogenous Dvl2 was immunoprecipitated from cell lysates at different time points with anti-Dvl2 antibody, and coimmunoprecipated
HA-Custos was detected by blotting. Lysates were immunoblotted with indicated antibodies to check sample loading. (C) Dvl2 interacts with NLS1
and NLS2 of Custos. Flag-Dvl2 and HA-tagged Custos mutants were transfected into HEK-293T cells and the proteins were immunoprecipitated from cell lysates
with the indicated antibodies. (D and E) The Custos binding region on Dvl2 mapped to between PDZ and DEP domain of Dvl2. HA-Custos and myc-tagged or Flag-tagged mutants of Dvl2 were transfected into HEK-293T cells and subjected to immunoprecipitation assays. (F) Custos interacts with casein kinase 1 (CK1).
GST-Custos protein was purified and incubated with in-vitro translated CK1. (G) Custos is phosphorylated by CK1. In vitro kinase reactions using GST or GSTCustos
revealed Custos is a substrate for CK1, CREB is used as a positive control. Image published in: Komiya Y et al. (2014) Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |