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. 2008 Dec 18;37(3):832–848. doi: 10.1093/nar/gkn941

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© 2008 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — SRC-2 exerts a synergistic effect with PRMT1 on TR3 transactivation. (A) Effect of SRCs on TR3 transactivation. Increasing amount of SRCs, including SRC-1, SRC-2 and SRC-3, with or without PRMT1 were transfected into 293T cells. The reporter gene activity of NurRE was determined. (B) PRMT1 and SRC-2 enhanced the binding of TR3 to Cyclin D2 promoter. Myc-PRMT1, Myc-PRMT1 (XGX) and HA-SRC-2 were transfected into 293T cells as indicated. The binding of TR3 to Cyclin D2 promoter was determined by ChIP assay using anti-TR3 antibodies, and then quantified by qPCR. (C) Effect of PRMT1/SRC-2 on endogenous TR3 binding to the NurRE. Myc-PRMT1 and HA-SRC-2 were transfected into 293T cells as indicated. Nuclear extracts were prepared, with or without incubated with anti-TR3 antibody, and then subjected to the EMSA assay with the NurRE or NBRE as a probe. (D) Both PRMT1 and SRC-2 bound to TR3. 293T cells were transfected with Flag-TR3, HA-PRMT1 and HA-SRC-2. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were then analyzed by western blotting using anti-Flag antibody to show the interaction of TR3 with PRMT1 and SRC-2, respectively. (E) SRC-2 interacted with TR3 in vivo. 293T cells were lysated and immunoprecipitated with anti-TR3 antibodies. The pellets were subjected to western blotting using anti-SRC-2 antibody. IgG was set as a negative control. (F) SRC-2 but not PRMT1 enhanced TR3-PRMT1 interaction. 293T cells were transfected with different plasmids as indicated. After transfection, cells lysates were immuoprecipitated with anti-Myc or anti-Flag antibodies, and then the immunoprecipitates were subjected to western blotting. (G) Determination of SRC-2-binding sites in TR3. 293T cells were transfected with HA-SRC-2 and different Flag-TR3 deletion constructs as indicated in Figure 2A. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were analyzed by western blotting using anti-Flag antibody for TR3 proteins. (H) Synergistic effect of SRC-2 with PRMT1 on TR3 transactivation. 293T cells were transfected with increasing amount of PRMT1, together with GAL4 reporter gene and GAL4-TAD or GAL4-LBD in the absence or presence of SRC-2 as indicated. The luciferase activity was then determined.

Figure 3. — SRC-2 exerts a synergistic effect with PRMT1 on TR3 transactivation. (A) Effect of SRCs on TR3 transactivation. Increasing amount of SRCs, including SRC-1, SRC-2 and SRC-3, with or without PRMT1 were transfected into 293T cells. The reporter gene activity of NurRE was determined. (B) PRMT1 and SRC-2 enhanced the binding of TR3 to Cyclin D2 promoter. Myc-PRMT1, Myc-PRMT1 (XGX) and HA-SRC-2 were transfected into 293T cells as indicated. The binding of TR3 to Cyclin D2 promoter was determined by ChIP assay using anti-TR3 antibodies, and then quantified by qPCR. (C) Effect of PRMT1/SRC-2 on endogenous TR3 binding to the NurRE. Myc-PRMT1 and HA-SRC-2 were transfected into 293T cells as indicated. Nuclear extracts were prepared, with or without incubated with anti-TR3 antibody, and then subjected to the EMSA assay with the NurRE or NBRE as a probe. (D) Both PRMT1 and SRC-2 bound to TR3. 293T cells were transfected with Flag-TR3, HA-PRMT1 and HA-SRC-2. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were then analyzed by western blotting using anti-Flag antibody to show the interaction of TR3 with PRMT1 and SRC-2, respectively. (E) SRC-2 interacted with TR3 in vivo. 293T cells were lysated and immunoprecipitated with anti-TR3 antibodies. The pellets were subjected to western blotting using anti-SRC-2 antibody. IgG was set as a negative control. (F) SRC-2 but not PRMT1 enhanced TR3-PRMT1 interaction. 293T cells were transfected with different plasmids as indicated. After transfection, cells lysates were immuoprecipitated with anti-Myc or anti-Flag antibodies, and then the immunoprecipitates were subjected to western blotting. (G) Determination of SRC-2-binding sites in TR3. 293T cells were transfected with HA-SRC-2 and different Flag-TR3 deletion constructs as indicated in Figure 2A. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were analyzed by western blotting using anti-Flag antibody for TR3 proteins. (H) Synergistic effect of SRC-2 with PRMT1 on TR3 transactivation. 293T cells were transfected with increasing amount of PRMT1, together with GAL4 reporter gene and GAL4-TAD or GAL4-LBD in the absence or presence of SRC-2 as indicated. The luciferase activity was then determined.