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. 2009 Dec 2;285(4):2333–2339. doi: 10.1074/jbc.M109.090522

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Interaction between TAK1 and PP6 is polyubiquitin-dependent. A, 293 cells were transfected with expression vectors for FLAG-tagged PP6 (FLAG-PP6) and HA-tagged ubiquitin (HA-Ub). Cells were lysed, and proteins were immunoprecipitated (IP) with anti-FLAG antibody (left panels) or anti-HA antibody (right panels). Immunoprecipitates and whole cell lysates were analyzed by immunoblotting with an anti-HA antibody and anti-FLAG. Asterisks indicate nonspecific bands. B, 293 cells were transfected with expression vectors for FLAG-tagged PP6 (FLAG-PP6) and HA-tagged K48R or K63R mutant ubiquitin. Cells were lysed, and proteins were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates and whole cell lysates were analyzed by immunoblotting with an anti-HA antibody and anti-FLAG. Asterisks indicate nonspecific bands. C, 293 cells were transfected with expression vectors for T7-tagged TAK1 (T7-TAK1), FLAG-tagged PP6 (FLAG-PP6), Myc-tagged ubiquitin (Myc-Ub), and HA-tagged TAB2 (HA-TAB2) wild-type (WT) or the CUE domain-lacking mutant (Δ). Cells were lysed, and FLAG-PP6 was immunoprecipitated with anti-FLAG antibody. Immunoprecipitates and whole cell lysates were analyzed by immunoblotting with anti-T7, anti-HA, anti-Myc, and anti-FLAG antibodies. D, cell lysates were immunoprecipitated with anti-T7 antibody. The immunoprecipitates and whole cell lysates were analyzed with anti-FLAG, anti-HA, anti-Myc, and anti-T7 antibodies.