Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 −/− MEFs harbor H3R2me2 hypomethylation and display growth defects. ( A ) Total cellular RNA was isolated from primary MEFs of the indicated genotype and subjected to semi-quantitative RT–PCR. The migration of the DNA fragments for PRMT6 and GAPDH is shown. The molecular weight marker is the 1 kb ladder and the gel was visualized using ethidium bromide. No RT denotes the absence of reverse transcriptase and is a control used to show that RNA was indeed amplified and not DNA. ( B ) Total cellular proteins from PRMT6 +/ + and PRMT6 −/− MEFs were separated by SDS–PAGE and subsequently immunoblotted with anti-PRMT6, anti-H3R2(me2a), anti-H3 and anti-α-Tubulin antibodies. ( C ) PRMT6 +/ + and PRMT6 −/− MEFs growing in log phase were trypsinized, fixed in 75% MeOH, stained with PI and analyzed by flow cytometry.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Isolation, Quantitative RT-PCR, Migration, Molecular Weight, Marker, Reverse Transcription, Control, Amplification, SDS Page, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 associates with the promoter region of Trp53 to regulate H3R2 methylation. ( A ) A schematic representation of the murine Trp53 gene with the primers used for ChIP analysis. The transcription start site is shown with the numbering upstream of this site. ChIP-qPCR analysis was performed using wild-type MEFs and the immunoprecipitated DNA enriched using anti-PRMT6, -PRMT1, -CARM1 or -PRMT5 antibodies was normalized with an internal IgG control. ( B ) ChIP-qPCR analysis using anti-PRMT6 antibodies from PRMT6 +/ + and PRMT6 −/− primary MEFs at −1322 of the Trp 53 promoter is shown. PRMT6 occupancy was normalized to an IgG control. ( C ) The presence of H3R2(me2a), H3K4(me3), H3R26(me2a) and H3R17(me2a) at the −1322 upstream region of Trp 53 promoter was determined using ChIP-qPCR analysis. The values for the histone marks were normalized to total histone H3 levels. ( D ) SA-β-Gal staining of PRMT6 −/− , p53 −/− , PRMT6 −/− ; p53 −/− and wild-type primary MEFs is shown. The numbers represent the percentage of SA-β-Gal positive cells. ( E ) Whole-cell extracts obtained from PRMT6 −/− ; p53 −/− MEFs or controls were analyzed by immunoblotting using anti-p53, -PML, -PRMT6 antibodies. Anti-α-tubulin was used as a loading control. Molecular mass markers for PML isoforms are shown on the right.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Methylation, ChIP-qPCR, Immunoprecipitation, Control, Staining, Western Blot