histone h3 asym dimethyl arg2 antibody  (Novus Biologicals)

Journal: Frontiers in Cell and Developmental Biology

Article Title: F-Box Protein FBXW17-Mediated Proteasomal Degradation of Protein Methyltransferase PRMT6 Exaggerates CSE-Induced Lung Epithelial Inflammation and Apoptosis

doi: 10.3389/fcell.2021.599020

Figure Lengend Snippet: FBXW17/protein arginine N-methyltransferase 6 (PRMT6) signaling involves in cigarette smoke extract (CSE)-induced lung epithelial inflammation and apoptosis. (A) Vector, FBXW17-FLAG plasmids, scramble small hairpin RNA (shRNA), and FBXW17 shRNA were transfected into MLE12 cells separately. Annexin V and PI kits were used to detect the rate of apoptotic cells. Before detection, cells were treated with CSE for 6 h. The “percentage of cells ± SD” values were shown in all the quadrants of flow cytometry. (B) The plotted data of apoptosis in each group are presented. (C) MLE12 cells were transfected with either overexpressed or knockdown plasmids of FBXW17. Before collection, the cells were treated with 5% CSE for 6 h. Cell lysate was conducted with immunoblotting with indicated antibodies. (D) The plotted PRMT6, H3R2me2a, and Bcl-2 protein expression were shown. (E) The relative protein expression of H3K4me3, Bax, TNF-α, IL-1β, and COX-2 were presented. Data were represented by n = 3 separate experiments. The graph shows mean ± SD. “ ∗ ” denotes p < 0.05 between indicated groups.

Article Snippet: H3R2me2a (catalog no. NB21-1002) was purchased from Novus Biologicals (Littleton, CO, United States).

Techniques: Plasmid Preparation, shRNA, Transfection, Flow Cytometry, Knockdown, Western Blot, Expressing

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: Antibodies used were the following: α-IgG (Vector Laboratories, I-2000); α-PRMT6 (Bethyl laboratories, A300-928 A); α-H3R2(me2a) (Novus Biologicals, NB21-1002); α-H3K4(me3) (Millipore, 17-614); α-H3R17(me2a) (Millipore, 07-214); α-H3R26(me2a) (Millipore, 07-215); α-H3 (Abcam, ab1791).

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: Antibodies used were the following: α-IgG (Vector Laboratories, I-2000); α-PRMT6 (Bethyl laboratories, A300-928 A); α-H3R2(me2a) (Novus Biologicals, NB21-1002); α-H3K4(me3) (Millipore, 17-614); α-H3R17(me2a) (Millipore, 07-214); α-H3R26(me2a) (Millipore, 07-215); α-H3 (Abcam, ab1791).

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