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. Author manuscript; available in PMC: 2009 Jun 11.
Published in final edited form as: Oncogene. 2008 Sep 15;27(58):7235–7247. doi: 10.1038/onc.2008.337

Figure 1. CKIγ2 constitutively and selectively interacts with Smad3.

Figure 1

(a) 293T cells were transfected with the indicated constructs. Cells were lysed in universal lysis buffer supplemented with protease and phosphatase inhibitors (ULB+) at 24 h post-transfection and anti-HA immunoprecipitation was performed. 2SD, S423/425D; KD, kinase-dead; WCL, whole-cell lysate. (b) The Flag-tagged, full-length Smad constructs were co-expressed with GFP-CKIγ2(WT). Anti-Flag immunoprecipitation was performed as in (a). The arrowhead indicates the heavy chain (hc) of the Flag antibody (M2). (c) Parental HaCaT cells were pretreated with 10 µM MG-132 for 1 h before treatment with or without 100 pm TGF-β for another 2 h. Endogenous CKIγ2 was precipitated with a goat polyclonal anti-CKIγ2 antibody (C-20), and endogenous Smad proteins were blotted with a monoclonal anti-Smad1/2/3 antibody. Co-precipitated Smad3 is indicated by an arrow. An irrelevant goat polyclonal antibody (IgG) was used as a negative control. (d) Wild-type Smad3 and CKIγ2 were co-expressed in MEFs for 20h. Cells were then treated with 100pm TGF-β for the indicated time course and then lysed for anti- Flag immunoprecipitation. CKIγ2, casein kinase 1 gamma 2; HA, hemagglutinin; MEFs, mouse embryonic fibroblasts; TGF-β, transforming growth factor-beta.