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Results: 5

1.
FIGURE 5.

FIGURE 5. From: Epigenetic Regulation of Transcriptional Activity of Pregnane X Receptor by Protein Arginine Methyltransferase 1.

PXR regulates PRMT1 subcellular localization as determined by immunofluorescence microscopy. Parental HepG2 and HT29 cells as well as the PXR stable transfectants PXR-HepG2 and PXR-HT29 were analyzed for PXR and PRMT1 subcellular localization by immunofluorescence microscopy.

Ying Xie, et al. J Biol Chem. 2009 April 3;284(14):9199-9205.
2.
FIGURE 4.

FIGURE 4. From: Epigenetic Regulation of Transcriptional Activity of Pregnane X Receptor by Protein Arginine Methyltransferase 1.

Recruitments of PRMT1, PXR, and changes of histone modifications in the CYP3A4 regulatory regions in response to PXR activation. PXR-HepG2 cells were treated with rifampicin (10 μm, 2 h). ChIP assay was performed to analyze the association of PXR, PRMT1, and changes of histone H4 acetylation and H4R3 methylation. Results were analyzed by quantitative real-time PCR. *, statistically significant difference (t test, p < 0.01). The data are the means ± S.D. of three independent results.

Ying Xie, et al. J Biol Chem. 2009 April 3;284(14):9199-9205.
3.
FIGURE 1.

FIGURE 1. From: Epigenetic Regulation of Transcriptional Activity of Pregnane X Receptor by Protein Arginine Methyltransferase 1.

Histone methyltransferase activity is associated with PXR. A, PXR-HepG2 cells were treated with rifampicin (10 μm, 2 h) and used to perform co-immunoprecipitation/HMT assay with anti-FLAG antibody. The precipitates and recombinant PRMT1 were subjected to HMT assay, respectively, with core histones as the substrates and [3H]SAM as the methyl donor. Methylated histones were analyzed by autoradiography. B, methylated H4 in A was subjected to N-terminal sequencing analysis. The radioactivity associated with Edman degradation fractions was determined by liquid scintillation counting. C, illustration of N-terminal sequences of histone H2A and H4 with the common “SGRGK” motif. D, substrate specificity comparison of the PXR-assocated HMT and recombinant PRMT1. Same molar pre-acetylated H4 N-terminal peptides (0.4 μg, 2 kDa) and recombinant H4 (2 μg, 11 kDa) (Upstate) were used as the substrates and [3H]SAM was used as the methyl donor. The pre-acetylated H4 peptides are 20-amino acid N-terminal peptides with K5, K8, K12, or K16 individually acetylated. Methylation was detected by autoradiography.

Ying Xie, et al. J Biol Chem. 2009 April 3;284(14):9199-9205.
4.
FIGURE 3.

FIGURE 3. From: Epigenetic Regulation of Transcriptional Activity of Pregnane X Receptor by Protein Arginine Methyltransferase 1.

PRMT1 is required for PXR transcriptional activity. A, PXR activity in PRMT1(-/-) ES cells. Gal4-driven luciferase reporter gene and Gal4-mPXR were transiently transfected into mouse PRMT1-null ES cells or wild-type ES cells. The transfected cells were treated with the receptor agonist PCN (10 μm, 24 h). Luciferase activity was determined by a luminometer. *, statistically significant difference (t test, p < 0.01). The data are the means ± S.D. of three independent results. B and C, the effect of siRNA knockdown of PRMT1 on PXR transcriptional activity. PXR-HepG2 cells were transfected with CYP3A4-luciferase. Two siRNAs targeting different sequences of PRMT1 (756–773 and 353–371) were used to knockdown PRMT1. Scrambled siRNA was used as the control (B and upper panel of C). The total PRMT1 protein expression was analyzed by Western blotting with PRMT1 antibody. Western blot with α-tubulin antibody was shown for loading control (C, lower panel). Lane 1, siPRMT1–11; lane 2, siPRMT1–28; lane 3, control. The reporter gene expression was measured by luciferase assay. *, statistically significant difference (t test, p < 0.01). The data are the means ± S.D. of three independent results.

Ying Xie, et al. J Biol Chem. 2009 April 3;284(14):9199-9205.
5.
FIGURE 2.

FIGURE 2. From: Epigenetic Regulation of Transcriptional Activity of Pregnane X Receptor by Protein Arginine Methyltransferase 1.

PRMT1 interacts with PXR in a ligand-dependent manner. A, PXR-HepG2 cells were treated with rifampicin (10 μm, 0, 30, 60, 90, 120 min) and subjected to co-immunoprecipitation with anti-FLAG antibody-coupled beads. The precipitates were eluted with 3× FLAG peptide and analyzed by Western blotting with PRMT1 antibody. Anti-FLAG antibody blotting was used to show the equal loading of the samples. B, liver tissue from a VP16-hPXR transgenic mouse was homogenized in the Co-IP lysis buffer and co-immunoprecipitated with goat anti-PXR (lane 4) and rabbit anti-PRMT1 antibodies (lane 3). Goat IgG (lane 5) and rabbit IgG (lane 2) were used as negative controls. 1:10 lysate was loaded as the input control (lane 1). Precipitates were analyzed by Western blotting with PRMT1 antibody. C, CV-1 cells were transfected with the bait plasmid, pBIND-PXR, and the reporter pG5-luc vector, with cotransfection of the prey plasmid pACT-PRMT1 or blank pACT plasmid. Six hours after transfection, cells were treated with rifampicin (10 μm) or vehicle for an additional 48 h. The interaction was characterized by luciferase activity. *, statistically significant difference (t test, p < 0.01). The data are the means ± S.D. of three independent results. D, mapping of the interactive domains of PXR with PRMT1 by GST pull-down assay. Various PXR fragments were fused with GST and the fusion peptides coupled with glutathione-Sepharose beads were incubated with radiolabeled PRMT1. The precipitated complexes were analyzed by autoradiography following SDS-PAGE (middle panel). Upper panel, illustration of PXR fragments. Lower panel, loading control of the GST-fused PXR fragments (Coomassie Blue staining).

Ying Xie, et al. J Biol Chem. 2009 April 3;284(14):9199-9205.

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