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. 2012 Feb 29;287(16):12867–12878. doi: 10.1074/jbc.M111.307249

FIGURE 4.

FIGURE 4.

SIK forms protein complexes with Smad7 and Smurf2. A, TGFβ-induced Smurf2 mRNA expression is not dependent on de novo protein synthesis. Quantitative real-time RT-PCR analysis measuring Smurf2 mRNA levels with or without cycloheximide pretreatment for 1 h before stimulation with 5 ng/ml TGF-β for 1, 2, 4, 8, or 24 h is shown. The data are presented as in Fig. 1. B, co-precipitation of wild-type 6Myc-SIK and catalytically inactive Myc-Smurf2(CG) after immunoprecipitation of FLAG-Smad7 is shown. Total cell lysate (TCL) controls are shown. An asterisk indicates a nonspecific protein band. Note that the immunoprecipitation and total cell lysate proteins have been resolved on two different gels as illustrated by the size markers. C, shown is co-precipitation of endogenous Smad7, Smurf2, and ALK5 after immunoprecipitation (IP) of endogenous SIK in the absence (−) or presence (+) of TGFβ1 stimulation for 16 h. Immunoprecipitation with an unrelated immunoglobulin (IgG Ctrl) served as negative control. Total cell lysate controls are also shown, and GAPDH serves as the protein loading control. D, shown is co-precipitation of wild-type or kinase-dead (K56R) 6Myc-SIK and wild-type or catalytically inactive Myc-Smurf2(CG) after immunoprecipitation of wild-type FLAG-Smad7. Note that the two top immunoblots represent anti-Myc blots at two different exposure times. The top, long exposure shows the 6Myc-SIK, and the second, short exposure shows the Myc-Smurf2. Lane numbers are duplicated at the top and bottom of the immunoblots for convenience.