Journal: Molecular oncology

Article Title: PELP1 oncogenic functions involve alternative splicing via PRMT6

doi: 10.1016/j.molonc.2013.12.012

Figure Lengend Snippet: Coordination of PELP1 and PRMT6 in alternative splicing. A,B) qRTPCR of VEGF isoforms 121, 165 and 189 normalized over total VEGF from ZR75shControl or ZR75shPELP1 cells transfected with either siControl or siPRMT6. Relative expression is from triplicates normalized to actin. C,D) Isoform specific qRTPCR of inclusion or skipping of exon 19 of NIN from ZR75shControl or ZR75shPELP1 cells transfected with siControl or siPRMT6. Relative expression is from triplicates normalized to actin.

Article Snippet: Histone methyltransferase assay Recombinant PRMT6 (cat# 11313‐H18H, Sino Biological Inc.) and Histone H3 (cat# 14‐411, Millipore) were used for the histone methyltransferase assay.

Techniques: Transfection, Expressing

Journal: Molecular oncology

Article Title: PELP1 oncogenic functions involve alternative splicing via PRMT6

doi: 10.1016/j.molonc.2013.12.012

Figure Lengend Snippet: Effect of PRMT6 on PELP oncogenic functions. A) Immunoprecipitation of PELP1 or PRMT6 was performed from nuclear lysates of ZR75 cells grown in 8% FBS‐RPMI. Western blot analysis was performed for co‐immunoprecipitation of PRMT6 or PELP1 with IgG and Input as the controls. B) GST binding assay with bacterial purified PELP1‐GST deletions and recombinant PRMT6 with GST as the control. Western blot is shown for PRMT6 expression with PRMT6 input as the control and ponceau stain shows PELP1 input protein denoted by asterisk. C) An ERE‐luciferase gene reporter assay was performed with ZR75vector and ZR75PELP1 cells transfected with siControl or siPRMT6 and ERE‐luciferase in triplicate. Cells were starved of estrogen in 5% DCC media for 48 h and treated with estradiol 10−8 M for 12 h. Cells were lysed with passive lysis buffer and relative luciferase activity was measured by a luminometer. D) An MTT proliferation assay was performed with ZR75vector and ZR75PELP1 cells transfected with either control siRNA or PRMT6 siRNA. E) ZR75 and ZR75PELP1 cells were transfected with either control siRNA or PRMT6 siRNA and plated in triplicate in 6‐well plates for a colony formation assay. Colonies were stained by crystal violet and counted on day 14. F) MCF7PELP1shRNA and ZR75PELP1shRNA cells were transfected with either control siRNA or PRMT6 siRNA and plated in triplicate in 6‐well plates for a colony formation assay. Colonies were stained by crystal violet and counted on day 14.

Article Snippet: Histone methyltransferase assay Recombinant PRMT6 (cat# 11313‐H18H, Sino Biological Inc.) and Histone H3 (cat# 14‐411, Millipore) were used for the histone methyltransferase assay.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Purification, Recombinant, Expressing, Staining, Luciferase, Reporter Assay, Transfection, Lysis, Activity Assay, Proliferation Assay, Colony Assay

Journal: Molecular oncology

Article Title: PELP1 oncogenic functions involve alternative splicing via PRMT6

doi: 10.1016/j.molonc.2013.12.012

Figure Lengend Snippet: Regulation of PRMT6 activity by PELP1. A) Histone methyltransferase assay was performed with recombinant Histone H3, PRMT6 enzyme, purified PELP1 in buffer with S‐adenosyl methionine, estradiol and purified estrogen receptor alpha. Western blot analysis shows expression of H3R2me2a, PRMT6 and PELP1 with Total Histone H3 as the control. B) ChIP assay with ZR75shControl and ZR75shPELP1 cells starved of estrogen for 72 h and treated with estradiol for 30 min was performed with H3R2me2a or IgG. qRTPCR was done in triplicate for GREB1C. C) ChIP assay with ZR75shControl and ZR75shPELP1 cells starved of estrogen for 72 h and treated with estradiol for 30 min was performed with PRMT6 or IgG. qRTPCR was done in triplicate for GREB1C. D) Sequential ChIP of ZR75PELP1 cells grown in 2.5% DCC for 72 h and treated with estradiol 10−7 M for 30 min was done with PELP1, PRMT6 or IgG. qRTPCR was performed in triplicate for GREB1C. E) Immunohistochemistry of tissues of xenografts treated with siControl or siPELP1 liposomes probed for PRMT6 or H3R2me2a expression. Quantification of ten sections per group as percentage of positive cells over total cells is shown on right.

Article Snippet: Histone methyltransferase assay Recombinant PRMT6 (cat# 11313‐H18H, Sino Biological Inc.) and Histone H3 (cat# 14‐411, Millipore) were used for the histone methyltransferase assay.

Techniques: Activity Assay, HMT Assay, Recombinant, Purification, Western Blot, Expressing, Immunohistochemistry

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: PRMT6 coactivates SHRs in HeLa cells. ( A ) PRMT6 coactivates ERα transcriptional activity from an oestrogen response element linked to a minimal promoter. HeLa cells were co-transfected with an ERE-E1b-luciferase reporter along with expression vectors for ERα alone, or with PRMT6 or PRMT6 V86K/D88A as indicated. Cells were treated with vehicle (ethanol) or 10 −9 M E2 as indicated and tested for luciferase activity (see ‘Materials and Methods’ section). ( B ) PRMT6 coactivates ERβ transcriptional activity from the ERE-E1b-luciferase reporter. HeLa cells were co-transfected with an ERβ expression plasmid as in (A). ( C ) PRMT6 enhances the transcriptional activity of PR. HeLa cells were co-transfected with a PRE-E1b-luciferase reporter along with expression vectors for PR alone, or with PRMT6 or PRMT6 V86K/D88A mutant and treated with vehicle (ethanol) or 10 −8 M Pg as indicated. ( D ) PRMT6 enhances the transcriptional activity of GR. HeLa cells were co-transfected with PRE-E1b-luciferase reporter along with expression vectors for GR alone, or PRMT6 or PRMT6 V86K/D88A mutant and treated with vehicle (ethanol) or 10 −8 M Dex as indicated. ( E ) PRMT6 does not coactivate the RARα. HeLa cells were co-transfected with RARE-E1b-luciferase reporter along with expression vectors for RARα alone, or with PRMT6 or PRMT6 V86K/D88A mutant and treated with vehicle (ethanol) or 10 –7 M RA as indicated. ( F ) PRMT6 does not coactivate PPARγ transcriptional activity. HeLa cells were co-transfected with a PPAR-TK-luciferase reporter along with expression vectors for PPARγ alone, or with PRMT6 or PRMT6 V86K/D88A mutant and treated with vehicle (ethanol) or with 10 –5 M Cig as indicated. Each data point represents the mean and standard deviation (SD) of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments. * P < 0.001; NS, no significant change.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Mutagenesis, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: PRMT6 synergistically coactivates ERα transcriptional activity in the presence of SRC-1. ( A ) CV-1 cells were transiently co-transfected with an ERE-E1b-luciferase reporter, 5-ng expression vector for ERα, along with expression vectors for PRMT6 or SRC-1 as indicated. Following treatment with vehicle (ethanol) or 10 –9 M E2, cells were assayed for luciferase activity. Numbers above bars show fold increase in luciferase activity compared to transfection with ERα alone and 10 –9 M E2 stimulation. Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments. ( B ) Mammalian-2-hybrid analysis demonstrates that PRMT6 interacts with full-length SRC-1. HeLa cells were co-transfected with pG5-E1b-luciferase reporter plasmid along with expression vectors for the Gal4 DNA-binding domain alone (Gal4), Gal4-SRC-1 chimera, the VP16 transcriptional activation domain alone (VP16) or VP16-PRMT6 chimera as indicated. Cell extracts were tested for luciferase activity. Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments. * P < 0.001. ( C ) GST pull-down assays were used to test the ability of GST-PRMT6 to interact with full length or fragments of 35 S-radiolabelled SRC-1. GST alone and in vitro translated 35 S-radiolabelled luciferase served as negative controls. A schematic representation of the SRC-1 protein fragments used in the assays is shown in the panel, with amino acid positions of the SRC-1 protein or SRC-1 protein fragments indicated. The major functional domains of SRC-1 are indicated, bHLH/PAS, NR Boxes, AD1 and AD2. ( D ) Recruitment of PRMT6 to oestrogen response elements (EREs) located in promoter regions of the GREB1 gene. Following 0-, 15- and 45-min treatment of MCF-7 cells with 10 −9 M E2, recruitment of PRMT6 to EREs in the GREB1 promoter and to a site downstream of the GREB1 transcriptional start site (+6 kb) was determined by chromatin immunoprecipitation as detailed in ‘Materials and Methods’ section. Results show the average and SD of four independent experiments. ( E ) Recruitment of PRMT6 to an oestrogen-receptor-binding enhancer region of the PR gene. Following 0-, 15- and 45-min treatment of MCF-7 cells with 10 −9 M E2, recruitment of PRMT6 to a known oestrogen-receptor-binding enhancer region of the PR gene (PR ERE) and to a site downstream of the PR gene transcriptional start site (+5 kb) was determined by chromatin immunoprecipitation as detailed in ‘Materials and methods’ section. Results show the average and SD of four independent experiments.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Binding Assay, Activation Assay, In Vitro, Functional Assay, Chromatin Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: PRMT6 and CARM1 synergistically coactivate ERα transcriptional activity in the presence of SRC-1. CV-1 cells were co-transfected with an ERE-E1b-luciferase reporter, 0.15-ng expression vector for ERα, and combinations of expression vectors for PRMT6, CARM1, PRMT1 or SRC-1 as indicated. Following oestrogen treatment, cells were assayed for luciferase activity. Numbers above bars show fold increase in luciferase activity upon 10 −9 M E2 stimulation. Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: Knockdown of PRMT6 expression disrupts oestrogen signalling. ( A ) Western blot showing PRMT6, CARM1 and β-tubulin expression in MCF-7 cells following transfection with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1) and treatment with or without 10 −9 M E2 for 12 h as indicated. ( B ) Graphical representation of PRMT6 expression levels of western blot shown in (A). ( C ) Q-RT-PCR analysis of PRMT6 RNA levels in MCF-7 cells following transfection by control siRNA or siRNA targeting PRMT6 and treatment with or without 10 −9 M E2 for 12 h as indicated. ( D ) CARM1 expression as detected in (C). ( E ) PRMT1 expression as detected in (C). ( F ) GREB1 expression levels following PRMT6 knockdown. RNA was analysed by Q-RT-PCR for expression of GREB1 as in (C). ( G ) PR expression levels following PRMT6 knockdown. PR levels were examined as in (C). Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of two independent experiments. * P < 0.005; NS, no significant change compared to treatment with control siRNA.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Knockdown, Expressing, Western Blot, Transfection, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: Knockdown of PRMT6 and CARM1 expression inhibits oestrogen-stimulated proliferation of breast cancer cells. ( A ) MCF-7 cells were transfected with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1), CARM1 (C1-siRNA-1) or both PRMT6 and CARM1 (P6-siRNA-1 and C1-siRNA-1). Following treatment with 10 −9 M E2, cell proliferation was determined by 3 H-thymidine incorporation. ( B ) MCF-7 cells were transfected as in (A) and cell proliferation was determined by 3 H-thymidine incorporation in the absence of 10 −9 M E2. Each data point represents the mean and SD of results from eight individual cultures. Results are shown from a single experiment, which is representative of two independent experiments. * P < 0.001.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Knockdown, Expressing, Transfection, Control

Journal: Nucleic Acids Research

Article Title: Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

doi: 10.1093/nar/gkp1203

Figure Lengend Snippet: PRMT6 regulates alternative splicing of endogenous VEGF and Syk. ( A ) Schematic representation of the major spliced products of the VEGF gene (not to scale). ( B ) Effect of PRMT6 knockdown on alternative splicing of VEGF. MCF-7 cells were transfected either with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1) and treated either with or without 10 −9 M E2 for 12 h as indicated. RNA was harvested from the cells and the relative expression level of each spliced isoform was determined by Q-RT-PCR analysis as detailed in ‘Materials and Methods’ section. ( C ) The relative VEGF 189:VEGF 165 ratio was obtained by dividing the relative VEGF 189 cDNA level by the relative VEGF 165 cDNA level for each experimental condition. ( D ) Schematic representation of the major alternatively spliced products of the endogenous Syk gene (not to scale). ( E ) Representation of the RHCglo-Syk-Exon 7 minigene construct, not to scale. ( F ) Effect of PRMT6 knockdown on splicing of endogenous Syk. MCF-7 cells were transfected either with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1) and treated either with or without 10 –9 M E2 for 12 h as indicated. RNA was harvested from the cells and Syk splicing was determined by reverse transcriptase-PCR. The relative Syk[L]:Syk[S] ratio was obtained by dividing the relative Syk[L] cDNA level by the relative Syk[S] cDNA level for each experimental condition. An autoradiograph of the radiolabeled Syk and β-2-microglobulin reverse transcriptase-PCR products from a representative experiment is shown. Lane 1; radiolabelled 100-bp DNA marker; lane 2; Ctrl-siRNA without 10 −9 M E2; lane 3; Ctrl-siRNA with 10 −9 M E2, lane 4; P6-siRNA-1 without 10 −9 M E2; lane 5; P6-siRNA-1 with 10 −9 M E2. ( G ) Effect of PRMT6 on the splicing of an RHCglo-Syk-Exon 7 minigene. HeLa cells were transiently co-transfected with a RHCglo-Syk-Exon 7 minigene along with expression vectors for PRMT6, the PRMT6 V86K/D88A mutant or CARM1. RNA was harvested from the cells and Syk splicing determined by reversetranscriptase-PCR. The relative RHCglo-Syk-Exon 7 inclusion (Syk[I]): RHCglo-Syk-Exon 7 exclusion (Syk[E]) ratio was obtained by dividing the relative Syk[I] cDNA level by the relative Syk[E] cDNA level for each experimental condition. RT-PCR without reverse transcriptase did not produce any detectable PCR products (data not shown). A representative autoradiograph of the radiolabelled RHCglo-Syk-Exon 7 minigene products is shown. For all experiments, each data point represents the mean and SD of results from four transfected cultures (or six transfected cultures for the RHCglo-Syk-Exon 7 minigene experiment). Results shown are from a single experiment, which is representative of two independent experiments. * P < 0.005, # P < 0.05.

Article Snippet: Gene expression levels were determined by Q-RT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Assay on Demand Taqman primer/probes (Applied Biosystems); PRMT6 (Hs00250803_s1), CARM1/PRMT4 (Hs00406354_m1), PRMT1 (Hs01587651_g1), GREB1 (Hs00536409_m1), PR (Hs01556701_m1), ERα (Hs01046818_m1) and normalised to GAPDH (Cat#4326317E) and RPLP0 (Cat#4326314E) expression levels.

Techniques: Alternative Splicing, Knockdown, Transfection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Reverse Transcription, Autoradiography, Marker, Mutagenesis

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Lung function (Mean±SD)

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Control

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Lung function and histological examination of mouse lungs. (A) Raw and Cdyn were recovered after PRMT6 treatment; n=8 mice per group. (B) HE staining of lung slides (×200) in the control group (a), CSE group (b), CSE+Lenti-NC group (c), and CSE+Lenti-PRMT6 group (d); n=4 mice per group. MLI (C) and DI (D) were calculated in each group. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δp<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Staining, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Expression of epigenetic protein in mouse lung. (A) Western blot analysis showed that overexpressing of PRMT6 caused significant changes of downstream target; n=4 mice per group. C: control group, S: CSE group, N: CSE+Lenti-NC group, P: CSE+Lenti-PRMT6 group. Relative density of PRMT6 (B), H3R2me2a (C) and H3K4me3 (D) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δp<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Immunohistochemical detection of NF-κB/p65 molecule and its correlation with H3K4me3. (A) Activated NF-κB/p65 were transferred into the nucleus (×400); n=4 mice per group. Dark brown nuclear cells were positive cells, shown in the control group (a), CSE group (b), CSE+Lenti-NC group (c), and CSE+Lenti-PRMT6 group (d). The percentage of positive cells (B) and its correlation with H3K4me3 (C) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δ p<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Immunohistochemical staining, Control, Comparison

Journal: Tobacco Induced Diseases

Article Title: PRMT6 mediates inflammation via activation of the NF-κB/p65 pathway on a cigarette smoke extract-induced murine emphysema model

doi: 10.18332/tid/116413

Figure Lengend Snippet: Effect of PRMT6 on inflammation in mouse lung. (A) Western blot analysis showed that overexpression of PRMT6 decreased pro-inflammatory factors; n=4 mice per group. Relative density of TNF-α (B) and IL-1β (C) were performed. *p<0.05 in comparison to control group; # p<0.05 in comparison to CSE group; Δ p<0.05 in comparison to CSE+Lenti-NC group

Article Snippet: The antibodies were: PRMT6 #14641S and H3K4me3 #9727S, (Cell Signaling Technology, USA); H3R2me2a ab8046, (Abcam, USA); TNF-α Sc-8301 and IL-1β Sc-7884 (Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Over Expression, Comparison, Control

Journal:

Article Title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression

doi: 10.1128/JVI.79.1.124-131.2005

Figure Lengend Snippet: (A) Arginine methylation of Tat by PRMT6. Histidine-tagged recombinant Tat (HIS-Tat) was incubated with [methyl-3H]S-adenosyl-l-methionine in the absence (lanes 1 and 5) and presence of recombinant GST-CARM1 (lanes 2 and 6), GST-PRMT6 (lanes 3 and 7), and GST-PRMT7 (lanes 4 and 8). The proteins were separated by SDS-PAGE, and the gels were stained with Coomassie blue. The incorporation of 3H label on Tat was visualized by fluorography. The migration positions of the GST-PRMTs and HIS-Tat are indicated to the left of the gel. (B) In vivo methylation assay of Tat. HA epitope-tagged Tat (HA-Tat) was transfected with either empty pVAX vector or vector expressing PRMT6 or methyltransferase-inactive PRMT6 (VLD-KLA). The transfected cells were metabolically labeled with l-[methyl-3H]methionine. HA-Tat was immunopurified, bound HA-Tat was resolved by SDS-PAGE, and labeled Tat was detected by fluorography. HA-Tat was also transfected alone, and the cells were incubated with l-[35S]methionine to ensure that protein synthesis was indeed inhibited (lane 4). (C) PRMT6 methylates the ARM. A schematic diagram of Tat with its cysteine-rich core (Cys-rich peptide Tat 25-39 [negative control]) and ARM (peptide 49-63). The sequences of the TAT peptides used for methylation are shown. The peptides were incubated with [methyl-3H]S-adenosyl-l-methionine in the presence of recombinant GST-PRMT6, and 3H incorporation was visualized by SDS-PAGE followed by fluorography.

Article Snippet: Anti-PRMT6 antibodies were purchased from IMGENEX (San Diego, Calif.).

Techniques: Methylation, Recombinant, Incubation, SDS Page, Staining, Migration, In Vivo, Transfection, Plasmid Preparation, Expressing, Metabolic Labelling, Labeling, Negative Control

Journal:

Article Title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression

doi: 10.1128/JVI.79.1.124-131.2005

Figure Lengend Snippet: Interaction of Tat with PRMT6. HeLa cells were transfected with expression vectors encoding HA-Tat and myc-PRMT6. The cells were lysed, and an aliquot was retained as total cell lysate (TCL). Cell extracts were incubated with either control immunoglobulin G (IgG) or with anti-HA or anti-Myc antibodies as indicated. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-Myc (lanes 1 to 3) and anti-HA (lanes 4 to 6) antibodies. The migration of myc-PRMT6 and HA-Tat is shown. The bands at ∼60 kDa are the heavy chains of the antibodies. The bands in lane 4 above 55 kDa are often observed with our anti-HA (12CA5) antibodies and are nonspecific.

Article Snippet: Anti-PRMT6 antibodies were purchased from IMGENEX (San Diego, Calif.).

Techniques: Transfection, Expressing, Incubation, SDS Page, Migration

Journal:

Article Title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression

doi: 10.1128/JVI.79.1.124-131.2005

Figure Lengend Snippet: PRMT6 expression inhibits Tat-mediated transactivation of the HIV-LTR promoter. HEK293T cells were transfected with the HIV LTR luciferase reporter plasmid along with expression vectors for HA-Tat, PRMT6 (A), and methylase-inactive PRMT6:VLD-KLA (B) or PRMT6 siRNA (C) as indicated. In panels A and B, the amount (0.5 and 1.0 μg) of PRMT6 or PRMT6:VLD-KLA is indicated by the thickness of the triangle below the bar. PRL-TK was included to control for transfection efficiency. The transfected cells were lysed 48 h after DNA transfection and assayed for luciferase activity, which was normalized to the activity of renilla. The luciferase activity without Tat was normalized to 1, and Tat induction is shown as fold induction. The results shown represent the mean ± standard error of the mean from three separate experiments for each bar (n > 9).

Article Snippet: Anti-PRMT6 antibodies were purchased from IMGENEX (San Diego, Calif.).

Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal:

Article Title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression

doi: 10.1128/JVI.79.1.124-131.2005

Figure Lengend Snippet: Reduced PRMT6 expression enhances HIV-1 LTR expression. HeLa MAGI cells were transfected with mock siRNA or PRMT6 siRNA. After 48 h, the cells were lysed and the mRNA (A) and protein (B) extracts were analyzed by RT-PCR and immunoblotting with anti-PRMT6 or anti-Sam68 antibodies. The presence (+RT) or absence (−RT) of reverse transcriptase is indicated above the lanes. gapdh, glyceraldehyde-3-phosphate dehydrogenase. (C) HeLa MAGI cells containing a stably integrated copy of HIV-1 LTR-β-galactosidase and expressing the CD4 receptor were transfected with mock siRNA or PRMT6 siRNA. At 24 h posttransfection, the cells were infected with 2 or 10 ng of T-tropic HIV-1. The cells were incubated for 48 h, fixed, and stained with X-Gal. The bars represent the number of blue cells counted in three or four nonoverlapping fields.

Article Snippet: Anti-PRMT6 antibodies were purchased from IMGENEX (San Diego, Calif.).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Reverse Transcription, Stable Transfection, Infection, Incubation, Staining

Journal:

Article Title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression

doi: 10.1128/JVI.79.1.124-131.2005

Figure Lengend Snippet: HIVp24 production is increased with the knockdown of PRMT6 and decreased with PRMT6 overexpression. HEK293T cells were transfected with the BH10 proviral HIV-1 plasmid along with mock siRNA, PRMT6 siRNA, or myc-PRMT6 expression vector. Forty-eight hours after transfection, the knockdown of PRMT6 was examined by immunoblotting with anti-PRMT6 antibodies (A) or anti-Myc antibodies (B). The same membranes were also immunoblotted with anti-Sam68 antibodies to verify equivalent loading. (C) HIV-1 production was assessed by measuring the level of HIV p24 present in the supernatant of the transfected cells by an ELISA after transfection with PRMT6 siRNA or expression vectors. The results shown represent the mean ± standard error of the mean from three separate experiments (n > 6).

Article Snippet: Anti-PRMT6 antibodies were purchased from IMGENEX (San Diego, Calif.).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay