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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 5. — TR3 attenuated PRMT1-induced methylation of STAT3 and Sam68. (A) TR3 inhibited the methylation activity of PRMT1 on a broad spectrum of substrates. Myc-PRMT1, with increasing amount of Flag-TR3 or TR3/D2, was transfected into 293T cells. Cell lysates were prepared and then subjected to western blotting. The methylation of proteins was detected by anti-dimethyl-Arginine asymmetric antibody (ASYM24). AdOx was used as a positive control. (B, D) TR3 attenuated PRMT1-induced methylation of STAT3. Flag-STAT3, siPRMT1, Myc-TR3, TR3/D2 and si-TR3 were transfected into 293T cells as indicated, and then immunoprecipitated with anti-Flag antibody. The methylation of STAT3 was determined with anti-dimethyl-arginine asymmetric antibody (ASYM24). AdOx treatment was set as a control. (C, E) TR3 abolished PRMT1-induced methylation of Sam68. Myc-Sam68, siPRMT1, Flag-TR3, TR3/D2 and si-TR3 were transfected into 293T cells as indicated. The methylation of Sam68 was determined. (F) Effect of TR3 on PRMT1-induced transactivation of STAT3. Increasing amount of PRMT1 or its mutant PRMT1(XGX), together with Flag-STAT3 or Flag-TR3 were transfected into 293T cells. After transfection, cells were treated with IL6 (10 ng/ml) for 30 min. The luciferase activity of APRE reporter gene was determined. *P < 0.05 by two-tailed Student's t-test. (G) Effect of TR3 on localization of Sam68. Myc-Sam68 with or without GFP-TR3 was transfected into 293T cells. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting with anti-Myc antibody. Tubulin and Lamin A were used to quantify the amount of protein loaded in the cytoplasm and nucleus. (H) Confocal analysis of Sam68 subcellular location regulated by TR3 and its deletion mutant. Myc-Sam68, GFP-TR3 and TR3/D2 were transfected into 293T cells at different combinations as indicated. Cells were immunostained by anti-Myc antibody followed by Texas Red-conjugated secondary antibody to detect Sam68. Stained cells were visualized with the confocal microscope. AdOx was used as a positive control.

Figure 5. — TR3 attenuated PRMT1-induced methylation of STAT3 and Sam68. (A) TR3 inhibited the methylation activity of PRMT1 on a broad spectrum of substrates. Myc-PRMT1, with increasing amount of Flag-TR3 or TR3/D2, was transfected into 293T cells. Cell lysates were prepared and then subjected to western blotting. The methylation of proteins was detected by anti-dimethyl-Arginine asymmetric antibody (ASYM24). AdOx was used as a positive control. (B, D) TR3 attenuated PRMT1-induced methylation of STAT3. Flag-STAT3, siPRMT1, Myc-TR3, TR3/D2 and si-TR3 were transfected into 293T cells as indicated, and then immunoprecipitated with anti-Flag antibody. The methylation of STAT3 was determined with anti-dimethyl-arginine asymmetric antibody (ASYM24). AdOx treatment was set as a control. (C, E) TR3 abolished PRMT1-induced methylation of Sam68. Myc-Sam68, siPRMT1, Flag-TR3, TR3/D2 and si-TR3 were transfected into 293T cells as indicated. The methylation of Sam68 was determined. (F) Effect of TR3 on PRMT1-induced transactivation of STAT3. Increasing amount of PRMT1 or its mutant PRMT1(XGX), together with Flag-STAT3 or Flag-TR3 were transfected into 293T cells. After transfection, cells were treated with IL6 (10 ng/ml) for 30 min. The luciferase activity of APRE reporter gene was determined. *P < 0.05 by two-tailed Student's t-test. (G) Effect of TR3 on localization of Sam68. Myc-Sam68 with or without GFP-TR3 was transfected into 293T cells. Nuclear and cytoplasmic fractions were prepared and subjected to western blotting with anti-Myc antibody. Tubulin and Lamin A were used to quantify the amount of protein loaded in the cytoplasm and nucleus. (H) Confocal analysis of Sam68 subcellular location regulated by TR3 and its deletion mutant. Myc-Sam68, GFP-TR3 and TR3/D2 were transfected into 293T cells at different combinations as indicated. Cells were immunostained by anti-Myc antibody followed by Texas Red-conjugated secondary antibody to detect Sam68. Stained cells were visualized with the confocal microscope. AdOx was used as a positive control.