CKIP-1 directly interacts with Smurf1 to promote Smurf1 degradation.

<p>DF-1 chicken fibroblasts were transfected with the indicated constructs and then immunostained using anti-FLAG and anti-V5 antibodies. (A) Localization of FLAG-tagged CKIP-1 and V5-tagged Smurf1 alone. (B) Schematics for CKIP-1 FLAG constructs highlighting the membrane-binding PH domain (red) and Smurf1-binding LZ (blue) followed by a C-terminal FLAG tag (yellow). (C) Full-length CKIP-1 FLAG was cotransfected with full-length V5-Smurf1 or V5-Smurf1 C700A dominant-negative mutant. In addition, CKIP-1 FLAG truncation mutants were cotransfected with full-length V5-Smurf1, showing that the PH domain is required for efficient membrane localization, and the LZ domain is required for Smurf1 colocalization. Arrows indicate membrane localization. Scale bars represent 10 μm. (D) Chick embryos were electroporated at HH4 with V5-Smurf1 and CKIP-1 FLAG and then stained for V5 or FLAG at HH8 and imaged in cryosection. Scale bars represent 25 μm. (E) Western blot analysis of HH7 whole-embryo lysates electroporated with constructs indicated and probed for V5, pSmad1/5/8, and RFP. Bar graphs represent normalized V5 and pSmad1/5/8 staining intensity means ± SEM from 3 replicates. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004425#pbio.2004425.s006" target="_blank">S1 Data</a>. <i>P</i> values from two-tailed Student <i>t</i> test. CKIP-1, casein kinase interacting protein 1; HH, Hamburger-Hamilton stage; LZ, leucine zipper motif; NC; neural crest domain; NS, not significant; PH, pleckstrin homology; pSmad1/5/8, phospho-Smads 1/5/8; RFP, red fluorescent protein; Smurf1, Smad ubiquitin regulatory factor 1.