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. 2007 Dec 17;179(6):1123–1131. doi: 10.1083/jcb.200703166

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Copyright © 2007, The Rockefeller University Press

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Figure 3. — PKD2 and PKD3 dimerize and transphosphorylate. (A) Specificity of PKD2 and PKD3 antibodies. Pure recombinant Flag-PKD2 and GST-PKD3 were Western blotted with anti-PKD2 and -PKD3 antibodies. Anti-PKD2 antibody recognizes PKD2 (lane 2) and anti-PKD3 antibody recognize PKD3 (lane 3). (B) PKD2 and PKD3 interact. PKD2 or PKD3 was immunoprecipitated from HeLa cell lysates with specific antibodies and the precipitates blotted with anti-PKD2 antibody (lanes 1 and 2) or PKD3 antibody (lanes 3 and 4). (C) Exogenously expressed PKD2 and PKD3 interact. GST (lane 1) or GST-PKD2 (lanes 2 and 3) was coexpressed with Flag-HRP (lane 2) or FLAG-PKD3 (lanes 1 and 3) in HeLa cells. The cells were immunoprecipitated with anti-Flag antibody (top left) or anti-GST antibody (top right) and Western blotted with anti-GST or anti-Flag antibody, respectively. To verify that each tagged protein was expressed and immunoprecipitated, the Flag and the GST precipitates were respectively blotted with anti-Flag (bottom left) and anti-GST (bottom right). (D) PKD2 and PKD3 interact directly. Pure recombinant GST-tagged proteins were incubated in vitro with pure recombinant Flag-tagged proteins. After GST pull-down, the precipitates were Western blotted with anti-Flag antibody, followed by anti-GST antibody. (E) PKD2 and PKD3 colocalize on PKD-KD tubes. HeLa cells were cotransfected with Flag-PKD2-KD and GST-PKD3-WT (top) or with Flag-PKD2-WT and GST-PKD3-KD (bottom). The cells were visualized by fluorescence microscopy with anti-PKD2 and anti-GST antibody. (F) PKD2 and PKD3 transphosphorylate. Pure recombinant GST-PKD2-WT was incubated alone (lane 1) or mixed with either Flag-PKD2-KD (lane 3) or Flag-PKD3-KD (lane 7) for in vitro kinase assays. Similarly, GST-PKD3-WT was incubated alone (lane 4) or mixed with Flag-PKD3-KD (lane 6) or Flag-PKD2-KD (lane 8). The lack of kinase activity of Flag-PKD2-KD and Flag-PKD3-KD is shown in lanes 2 and 5, respectively.