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. Author manuscript; available in PMC: 2014 Jan 4.
Published in final edited form as: Biochem J. 2013 Oct 15;455(2):10.1042/BJ20130281. doi: 10.1042/BJ20130281

Figure 3. S99 phosphorylation mediates binding of PAK4 to 14-3-3 proteins.

Figure 3

A. Hek293T cells were transfected with HA-tagged constitutively-active PKD1 (PKD1.CA). Endogenous LIMK was immunoprecipitated (α-LIMK). Following SDS-PAGE and transfer to nitrocellulose, samples were analyzed for co-immunoprecipitated endogenous PAK4 (α-PAK4) or 14-3-3 (α-14-3-3) and then probed for LIMK (α-LIMK). Additionally, lysates were analyzed by Western blotting for PKD1.CA expression (input control). B. Hek293T cells were co-transfected with FLAG-tagged PAK4 or PAK4.S99A mutant combined with vector or HA-tagged 14-3-3γ and vector or constitutively-active PKD1 (PKD1.CA), as indicated. PAK4 was immunoprecipitated from cell lysates using α-FLAG antibody. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting for co-immunoprecipitated 14-3-3γ (α-HA antibody). Samples were re-probed for total PAK4 (α-PAK4). Additionally, lysates were analyzed by Western blotting for PKD1 expression (α-PKD1) and 14-3-3γ (α-HA) expression. C. Hek293T cells were co-transfected with FLAG-tagged PAK4 or PAK4.S99A mutant combined with vector or HA-tagged 14-3-3γ and constitutively-active PKD1 (PKD1.CA), as indicated. 14-3-3γ was immunoprecipitated from cell lysates using α-HA antibody. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting for co-immunoprecipitated PAK4 (α-FLAG antibody). Samples were re-probed for total 14-3-3γ (α-HA). Additionally, lysates were analyzed by Western blotting for PKD1 expression (α-FLAG) and PAK4 (α-FLAG) expression. D. HeLa cells were co-transfected with FLAG-tagged PAK4 or PAK4.S99A mutant, HA-tagged 14-3-3γ and constitutively-active PKD1 (PKD1.CA), as indicated. Samples were subjected to immunofluorescence analysis in which PAK4 was detected by using α-FLAG antibody and Alexa Fluor 647 as secondary antibody and 14-3-3α was detected using an α-HA antibody and Alexa Fluor 568 as secondary antibody. E. In vitro kinase assays were performed with purified recombinant PKD1 and bacterially-expressed GST-tagged kinase-dead PAK4 (GST-PAK4.K350M), or GST-tagged kinase-dead PAK4 additionally mutated at S99A (GST-PAK4.K350M.S99A), as indicated. Following the kinase reactions, samples were separated on SDS-PAGE and transferred to nitrocellulose. A Far-Western blot was performed using recombinant HIS-tagged 14-3-3ζ. 14-3-3 protein bound to PKD1 and PAK4 was determined by immunostaining with anti-HIS antibody. Additional staining was performed to control equal input of purified PAK4 or PKD1 proteins.