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. 2011 Aug 9;286(39):34254–34261. doi: 10.1074/jbc.M111.259424

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — PKD regulates PAK4 activity in vivo. A, NMuMG cells (3 × 10⁵, 6-cm dish) were co-transfected with FLAG-tagged PAK4 and either vector control or constitutively active PKD1, PKD2, or PKD3, as indicated. PAK4 was immunoprecipitated (α-FLAG) and analyzed for phosphorylation at Ser-474 (α-pS474-PAK4). Blots were stripped and analyzed for total PAK4 (α-FLAG). Control blots were performed on lysates to determine expression of active PKD1 (α-PKD1 staining), PKD2 (α-FLAG staining), and PKD3 (α-GST staining). B, HeLa cells (0.5 × 10⁶, 6-cm dish) were treated with the PKD inhibitor CID755673 (20 μm) for 1 h. Endogenous PAK4 was immunoprecipitated (α-PAK4), and samples were analyzed for phosphorylation at Ser-474 (α-pS474-PAK4). Blots were stripped and analyzed for total PAK4 (α-PAK4).