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. 2018 Sep 13;20(10):1045–1058. doi: 10.1016/j.neo.2018.08.008

Figure 1.

Figure 1

TROY associates with PDZ-RhoGEF (PRG). (A) 293 cells were co-transfected with HA-tagged TROY plasmid and Myc-tagged PDZ-RhoGEF plasmid, and the lysates were immunoprecipitated with an anti-Myc antibody or mouse IgG. The immunoprecipitates were analyzed for the presence of PDZ-RhoGEF by immunoblot analysis. The expression level of Myc-tagged PDZ-RhoGEF and HA-tagged TROY in the cells is shown by immunoblotting of whole cell lysates (WCL). (B-D) The lysates of T98G, GBM10, and GBM43 cells were immunoprecipitated with an anti-TROY antibody or rabbit IgG. The immunoprecipitates were analyzed for the presence of PDZ-RhoGEF by immunoblot analysis. The expression level of PDZ-RhoGEF and TROY in the cells is shown by immunoblotting of WCL. (E) TROY does not co-immunoprecipitate with LARG in 293 cells. 293 cells were transfected with HA-tagged LARG or Myc-tagged TROY, or co-transfected with HA-tagged LARG and Myc-tagged TROY. Twenty-four hours after transfection, cells were lysed and immunoprecipitated with anti-Myc antibody. Immunoprecipitates or WCL were immunoblotted with the indicated antibodies. (F) Colocalization of TROY and PDZ-RhoGEF in T98G, GBM10, and GBM43 cells. Cells were stained and examined by confocal microscopy. TROY was visualized with Alexa Fluor 546–conjugated antibody, and PDZ-RhoGEF was visualized with Alexa Fluor 488–conjugated antibody. The colocalization of TROY (red) with PDZ-RhoGEF (green) appears as a yellow color in the merged image. Cells were counterstained with DAPI (blue) (bars = 10 μm). (G) 293 cells were co-transfected with AU1-tagged TROY, Myc-tagged PRG, and HA-tagged Pyk2, and 24 hours later, cells were lysed and immunoprecipitated with goat anti-Myc or goat anti-AU1 antibodies or goat IgG. Immunoprecipitates or WCL were immunoblotted (IB) with the indicated antibodies. (H) Left panel, 293 cells were transfected with HA-tagged LARG and/or FLAG-tagged Pyk2. Twenty-four hours later, the cells were lysed and immunoprecipitated with anti-FLAG antibody or mouse IgG. Immunoprecipitates or WCL were IB with anti-FLAG or anti-HA antibodies. Right panel, 293 cells were co-transfected with Myc-tagged PRG and FLAG-tagged WT Pyk2 or kinase-deficient (KD) Pyk2 for 24 hours. Cells were lysed and immunoprecipitated with anti-Myc antibody. Immunoprecipitates or WCL were IB with the indicated antibodies.