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. 2014 May 19;9(5):e98090. doi: 10.1371/journal.pone.0098090

Figure 4. RhoA increases PKD2/3 complex formation and PKD2 and PKD3 activities.

Figure 4

A: HeLa cells were transfected with vector control (pEBG; expresses GST), FLAG-tagged PKD2, GST-tagged PKD3 or GST-tagged active RhoA (pEBG-RhoA.CA), as indicated. Cells were lysed and PKD2 was immunoprecipitated (anti-FLAG). Samples were subjected to SDS-PAGE, transferred to nitrocellulose and immune-stained for co-immunoprecipitated PKD3 and RhoA.CA (anti-GST). After stripping samples were re-probed with anti-FLAG for PKD2. In addition, cell lysates were analyzed by Western blot (input control) for GST, GST-PKD3 or GST-RhoA.CA expression (anti-GST). B, C: HeLa cells were transfected with vector control (pEBG) or GST-tagged active RhoA (RhoA.CA), as indicated. Cells were lysed and endogenous PKD2 (B) or endogenous PKD3 (C) was immunoprecipitated (anti-PKD2 or anti-PKD3, respectively). Samples were subjected to SDS-PAGE, transferred to nitrocellulose and immunostained using anti-pS744/748 antibody that recognizes activation loop-phosphorylated PKD2 (pS706/710) or PKD3 (pS731/735). After stripping samples were re-probed with anti-PKD2 (B) or anti-PKD3 (C) antibodies. In addition, cell lysates were analyzed by Western blot (input control) for GST or GST-RhoA.CA expression (anti-GST). Experiments shown in A-C were performed at least three times with similar results.