anti h3r2me2a  (ABclonal Biotechnology)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

doi: 10.1186/s13046-024-03038-3

Figure Lengend Snippet: The Transcriptional Activation of YTHDF2 Mediated by PRMT6 Requires Its Methyltransferase Activity; Inhibiting This Activity Suppresses Glioma Cell Migration, Invasion, and EMT. A : Analysis of H3R2me2a, PRMT6, and YTHDF2 protein levels in LN229 and U251 cells treated with varying concentrations of EPZ020411 for 24 h. B : Assessment of YTHDF2 mRNA expression after 24-hour treatment with 20µM EPZ020411 in LN229 and U251 cells. C : Dual-luciferase reporter assays in LN229 or U251 cells treated with 20µM EPZ020411 for 24 h, with or without PRMT6 overexpression. D : Wound healing assay to evaluate cell migration in LN229 and U251 cells with or without EPZ020411 treatment. E-F : Transwell assays assessing cell migration and invasion with or without EPZ020411 treatment. Scale bar = 200 μm. G : Examination of EMT and Wnt-β-catenin pathway-related protein expression in LN229 and U251 cells treated with or without 20µM EPZ020411 for 24 h. H : Wild-type LN229 cells were intracranially implanted into nude mice followed by subcutaneous administration of EPZ or saline for three weeks to observe the effect of EPZ on in vivo tumorigenesis. Representative brain sections stained with H&E displayed xenograft tumors (upper panel, scale bar = 1.5 mm). In vivo invasion assays were conducted by examining the tumor margins in mouse brains (lower panel, scale bar = 100 μm). I : Tumor volumes for each group of mice were calculated. J : The relative invasive fingers of each tumor were calculated under a microscope by counting prominent and diffuse tumor tissues. K : Representative images of immunohistochemical staining for N-cadherin and E-cadherin in xenograft tumors from control and EPZ-treated groups of nude mice. Scale bar = 100 μm

Article Snippet: Anti-Histone H3R2me2a antibodies were from Genetex (USA, Cat#GTX54134) and PTM BIO (Hangzhou, China, Cat#PTM668).

Techniques: Activation Assay, Activity Assay, Migration, Expressing, Luciferase, Over Expression, Wound Healing Assay, Saline, In Vivo, Staining, Microscopy, Immunohistochemical staining, Control

Journal: International journal of oncology

Article Title: Inhibition of protein arginine methyltransferase 6 activates interferon signaling and induces the apoptosis of endometrial cancer cells via histone modification.

doi: 10.3892/ijo.2024.5620

Figure Lengend Snippet: Figure 2. PRMT6 inhibition suppresses EC cell proliferation accompanied by apoptosis with hypomethylation of H3R2me2a. (A) The mRNA expression levels of PRMT6 evaluated by using RT‑qPCR in PRMT6‑knockdown EC cell lines (HEC1B and HEC50B) are presented. (B) The protein expression levels of PRMT6 and H3R2me2a evaluated using western blotting in the same cell lines as in (A). (C) Cell viability assay in six types of EC cell lines (HEC1B, HEC50B, HEC265, Ishikawa, HEC151A, and HEC116). Cell cycle analysis (D) and Annexin assay (E) by flow cytometry in PRMT6‑knockdown EC cell lines (HEC1B and HEC50B). PRMT6, protein arginine methyltransferase 6; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction; H3R2me2a, histone H3R2 dimethylation. P<0.05 was considered to indicate a statistically significant difference. *P<0.01.

Article Snippet: Five types of anti‐H3R2me2a antibodies used for immunopre‐ cipitation did not work well, and their details were as follows: Anti‐histone H3 (asymmetric di methyl R2) antibody (2/25 μg chromatin; cat. no. ab175007; Abcam), anti‐dimethyl‐histone H3 (Arg2) antibody (10/100 μg chromatin; cat. no. 07‐585; MilliporeSigma), histone H3 [Asym‐dimethyl Arg2] antibody (10/100 μg chromatin; cat. no. NB21‐1002; Novus Biologicals, LLC), histone H3R2 dimethyl asymmetric (H3R2me2a) poly‐ clonal antibody (0.1/50 μg chromatin, 0.5/50 μg chromatin, 2/50 μg chromatin; cat. no. A‐3714‐050; Epigentek Group Inc.), anti‐histone H3 (asymmetric di methyl R2) antibody (0.1/50 μg chromatin, 0.5/50 μg chromatin, 1/50 μg chromatin; cat. no. ab194706; Abcam).

Techniques: Inhibition, Expressing, Western Blot, Viability Assay, Cell Cycle Assay, Annexin Assay, Flow Cytometry, Polymerase Chain Reaction

Journal: Nucleic acids research

Article Title: The arginine methyltransferase Prmt1 coordinates the germline arginine methylome essential for spermatogonial homeostasis and male fertility.

doi: 10.1093/nar/gkad769

Figure Lengend Snippet: Figure 4. Prmt1 depletion evokes an intrinsic compensatory increase of Prmt2 and Prmt6 responsible for the enhanced ADMA substra te methyla tion in testis. ( A ) Relati v e mRNA e xpression of PRMTs in testis from WT and Prmt1-uKO mice at indicated time (Supplementary Figure S3A), ( n = 3). *P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, Student’s t -test. ( B ) Relati v e mRNA e xpression of PRMTs in spleen from WT and Prmt1-uKO mice at indicated time (Supplementary Figure S3A) ( n = 3). *P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, Student’s t -test. ( C ) Immunoblotting of Prmt2 in WT and Prmt1-dKO mice at indicated time (Supplementary Figure S2C). Gapdh served as a loading control. ( D ) Quantification of normalized protein band intensities for ‘C’ ( n = 2). * P < 0.05 and ** P < 0.01. ( E ) Immunoblotting of Prmt5 in testis from WT and Prmt1-dKO mice a t indica ted time (Supplementary Figure S2C). ( F ) Quantification of normalized protein band intensities for ‘E’ ( n = 2). * P < 0.05. ( G ) Immunoblotting of Prmt6 in WT and Prmt1-dKO mice a t indica ted time (Supplementary Figure S2C). ( H ) Quantification of normalized protein band intensities for ‘G’ ( n = 2). *** P < 0.001 and **** P < 0.0001. ( I ) Immunoblotting of H3R2me2a, H4R3me2a in WT and Prmt1-uKO mice at indicated time (Supplementary Figure S3A). Staining by Coomassie brilliant blue (CBB) served as a loading control. ( J ) Quantification of normalized protein band intensities for ‘I’ ( n = 2). *** P < 0.001.

Article Snippet: The following primary antibodies were used for immunoblot: anti-Prmt1 (1:2000; M3008, Abmart), antiADMA (1:1000; 13522S, CST), anti-MMA (1:1000; 8711S, CST), anti-SDMA (1:1000; 13222S, CST), anti-Prmt2 (1:500; MG443642, Abmart), anti-Carm1 (1:500; 200135, ZEN BIO), anti-Prmt5 (1:1000; 381019, ZEN BIO), antiPr mt6 (1:1000; 383079, ZEN BIO), anti-Pr mt7 (1:3000; R25450, ZEN BIO), anti-c-Kit (1:1000; A0357, ABclonal Science), anti-H3 (1:1500; ET1701-64, HUABIO), anti-Gapdh (1:30 000; 60004-1-Ig, Proteintech ®), antiH4R3me2a (1:1500; 667, PTM BIO), anti-H4R3me2a (1:500; 39006, Acti v e Motif) and H3R2me2a (1:1000; A3714, EPIGENTEK).

Techniques: Western Blot, Control, Staining