rabbit polyclonal antisera against prmt5  (Santa Cruz Biotechnology)

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Loop formation requires Prmt5. ( A ) Western blot time course of Prmt5 protein levels over the time course of C3H10T1/2 cells differentiation. PI3K levels were monitored as a control. ( B – E ) C3H10T1/2 cells were infected with retroviruses containing either a control vector, wild-type Prmt5 or antisense Prmt5. (B) Western blots showing levels of Prmt5 in cells expressing empty vector, antisense and overexpressed Prmt5 96 h post-differentiation. GAPDH levels were monitored as a control. (C) Oil red O staining for cells 4 days post-differentiation. (D) Control vector and antisense Prmt5 expressing cells either collected just prior to differentiation (0 h) or 96 h later were tested by 3C for interactions between the PPARγ2 promoter and the −10 kb enhancer. (E) Control or cells overexpressing Prmt5 were differentiated for the specified times. Relative interaction frequencies for the interaction between the PPARγ2 promoter and the −10 kb enhancer were set to the value at the 0 h time point. All results are from three independent samples performed in triplicate. Error bars: Mean + SD (** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Western Blot, Control, Infection, Plasmid Preparation, Expressing, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Prmt5 binding at the PPARγ2 enhancer versus the promoter. ( A ) Samples were collected for ChIP from differentiated C3H10T1/2 cells infected with a retrovirus containing the control vector. ( B ) Samples were collected for ChIP from differentiated C3H10T1/2 cells infected with a retrovirus for Prmt5 to generate Prmt5 overexpression. ( C ) Overexpression of Prmt5 resulted in precocious PPARγ2 expression. RNA samples were taken at the specified time points. The level of PPARγ2 expression in cells expressing the control empty vector at time 0 was set to 1. Experiments from three independent samples were performed in triplicate and significance is based on values relative to the 0 h time point. (* P < 0.05, ** P < 0.01, by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Binding Assay, Infection, Control, Plasmid Preparation, Over Expression, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Knockdown of Prmt5 blocked binding of Brg1 and MED1 to the PPARγ2 locus. C3H10T1/2 cells were treated with one of two independent siRNAs for Prmt5 or a scrambled sequence control. ( A – C ) Oil Red O staining of cells treated with scrambled, Prmt5 siRNA Oligo 3 or Prmt5 siRNA oligo 5. ( D ) Western demonstrating Prmt5 knockdown. ( E – G ) Chromatin immunoprecipitation (ChIP) experiments were performed on C3H10T1/2 cells differentiated for 0, 12 and 96 h. DNA was immunoprecipitated with antibodies against (E) MED1, (F) Brg1 or (G) Prmt5. Top panels indicate binding at the PPARγ2 promoter while bottom panels indicate binding at the −10 kb enhancer. Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Analysis of three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Knockdown, Binding Assay, Sequencing, Control, Staining, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Overexpression of Prmt5 results in earlier recruitment of MED1 to the PPARγ2 promoter. C3H10T1/2 cells were infected with retrovirus containing either empty vector or Prmt5. ChIP experiments for MED1 were performed on cells differentiated for the indicated times. DNA immunoprecipitated by the MED1 antibody was amplified and quantified by real-time PCR for the ( A ) PPARγ2 promoter or ( B ) the −10 kb enhancer. Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Over Expression, Infection, Plasmid Preparation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Knockdown of MED1 blocked binding of Brg1 and Prmt5 to the PPARγ2 locus. C3H10T1/2 cells were treated with one of two independent siRNAs for MED1 or a scrambled control siRNA. ChIP experiments were performed on cells differentiated for the indicated times. DNA immunoprecipitated by antibodies against ( A ) MED1, ( B ) Brg1 or ( C ) Prmt5 was amplified and quantified by real-time PCR for the PPARγ2 promoter (top) and the PPARγ2 −10 kb enhancer (bottom). Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Knockdown, Binding Assay, Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: MED1 and Prmt5 are co-localized on the same regulatory sequences. Re-ChIP experiments were performed on C3H10T1/2 cells differentiated for 6 or 96 h. ( A ) Experiments analyzing binding to PPARγ2 promoter or enhancer sequences using a MED1 antibody followed by either IgG or a Prmt5 antibody for the second ChIP. ( B ) Experiments analyzing binding to PPAPγ2 promoter or enhancer sequences using a Prmt5 antibody followed by either IgG or a MED1 antibody for the second ChIP. Results are shown as % input rather than relative enhancement to allow better presentation of the IgG control. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Binding Assay, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Prmt5 mediates the formation of other DNA loops and inter-chromosomal interactions during adipogenic differentiation. ( A ) Time course of relative interaction frequency between the Ucp2 and Ucp3 loci. Samples of C3H10T1/2 cells were taken at the 0 h timepoint and at 3, 6, 12, 24 and 96 h following differentiation. ( B ) Control vector and antisense Prmt5 expressing C3H10T1/2 cells either collected prior to differentiation (0 h) or 96 h later were tested by 3C for interactions between the Ucp2 and Ucp3 loci. ( C ) Control vector and antisense Prmt5 expressing C3H10T1/2 cells either collected prior to differentiation (0 h) or 96 h later were used to determine expression levels of Ucp3 and Ucp2, normalized to levels of GAPDH, by quantitative PCR. Data are presented as fold induction relative to the expression at time 0, which was set to 1. ( D and E ) Interaction frequencies between the PPARγ2 promoter and the promoters of the adiponectin (AdipoQ) or perilipin 2 (Plin2) genes in C3H10T1/2 cells treated with scramble sequence (scr) siRNA or siRNAs targeting Prmt5 that were differentiated for the indicated times. All results are from three independent samples performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Control, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Sequencing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4

doi: 10.1093/nar/gkq896

Figure Lengend Snippet: Myogenic miRNA expression is compromised in Prmt5 antisense cell lines. ( A ) Immunoblot showing the Prmt5 and MyoD protein levels in vector- and MyoD-infected NIH 3T3 and Prmt5 antisense cell lines C1 and C12 at the onset of differentiation. The same blot was stripped and probed with PI3 Kinase (PI3K) antibody as a loading control. ( B ) qPCR analyses of primary transcripts of miR-1 and miR-133a upon MyoD-mediated differentiation of NIH 3T3 cells and the Prmt5 antisense cell lines, C1 and C12, along with the empty vector (EV) control. Expression levels were monitored 24 h post-differentiation. The expression of each miRNA in NIH 3T3 cells infected and differentiated with the empty vector retrovirus was normalized to 1. Results are the average of three independent experiments ± standard deviation.

Article Snippet: Western blots to detect Prmt5, MyoD and Carm1/Prmt4 were performed on whole cell extracts as described ( ) using rabbit polyclonal antisera against Prmt5 (Santa Cruz sc-22132), MyoD (Santa Cruz sc-32758) and Prmt4 (Millipore 09-818).

Techniques: Expressing, Western Blot, Plasmid Preparation, Infection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4

doi: 10.1093/nar/gkq896

Figure Lengend Snippet: Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. ( A and B ) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. ( C–F ) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.

Article Snippet: Western blots to detect Prmt5, MyoD and Carm1/Prmt4 were performed on whole cell extracts as described ( ) using rabbit polyclonal antisera against Prmt5 (Santa Cruz sc-22132), MyoD (Santa Cruz sc-32758) and Prmt4 (Millipore 09-818).

Techniques: Expressing, Infection, Standard Deviation, Plasmid Preparation, Control