Figure 3. Casein Kinase Iδ (CKIδ) negatively controlled NEDD4 stability.

(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with HA-NEDD4, Flag-β-TRCP1 and the indicated kinases. (B) IB analysis of WCL derived from 239T cells transfected with HA-NEDD4 and/or Myc-CKIδ together with Flag-WT–β-TRCP1 or Flag-R474A–β-TRCP1. (C) IB analysis of WCL and immunoprecipitates (IP) derived from HeLa cells transfected with HA-NEDD4 and Myc-tagged versions of the indicated CKI isoforms. (D) IB analysis of HeLa cells that were infected with shRNA specific for GFP or the indicated CKI isoforms, followed by selection with 1 μg/ml puromycin for three days to eliminate non-infected cells. (E) IB analysis of 293T cells treated with the CKI inhibitor D4476 at the indicated concentrations for 12 hours.