Recombinant PRMT7 and Antibody Validation

Anti-PRMT7 antibodies were purchased from Epicypher (#13-1009), Santa Cruz Biotechnology (SC9882) and Millipore (#07-639). Anti-Nanog (#61419), anti-Sox2 (#39823) and anti-H4R3me2s (#61187) antibodies were from Active Motif. Anti-Oct4 (#2840), anti-c-Myc (#5605) and anti-Klf4 (#4038) antibodies were from Cell Signaling. Anti-β-actin antibody (A5441) was from Sigma-Aldrich. Anti-H4R3me1 antibody (PA5-27065) was from Thermo Scientific. Anti-H3 antibody (ab1791) was from Abcam. Anti-mouse Argonaute 2 (Ago2) antibody was from Wako Chemicals (292-67301) and Cell Signaling (2897P). The cDNA constructs of human (OpenBiosystem) and mouse PRMT7 (OpenBiosystem) were cloned into pCDH1-EF1-IRES-GFP vector (System Biosciences) using the cloning sites EcoRI and NotI. Human PRMT7's catalytic mutant (m. PRMT7) was generated from pCDH1-EF1-PRMT7-IRES-GFP by mutating several residues (E144A, E153A, E478A and H644A) using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). These residues are likely located in arginine binding pockets according to crystal structures of PRMT1 and Caenorhabditis elegans PRMT7 (18 (link),19 (link)). Oligonucleotides used for cloning, ChIP-PCR, reverse-transcription (RT)-PCR, site-directed mutagenesis are listed in Supplementary Table S1.

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Lee S.H., Chen T.Y., Dhar S.S., Gu B., Chen K., Kim Y.Z., Li W, & Lee M.G. (2016). A feedback loop comprising PRMT7 and miR-24-2 interplays with Oct4, Nanog, Klf4 and c-Myc to regulate stemness. Nucleic Acids Research, 44(22), 10603-10618.

Publication 2016

Anti-Nanog anti-Sox2 anti-H4R3me2s Anti-Oct4 anti-c-Myc anti-Klf4 Anti-β-actin antibody ( Anti-H3 antibody (ab1791)

Actin Anti antibodies Anti antibody Antibodies Arginine Argonaute 2 Caenorhabditis elegans Catalytic Cdna Chip Ecori Human Ires Klf4 Mouse Oct4 Oligonucleotides Reverse transcription Site directed mutagenesis Sox2 Vector

Corresponding Organization : The University of Texas MD Anderson Cancer Center

Other organizations : Cornell University, Houston Methodist, Sungkyunkwan University, Samsung (South Korea), Baylor College of Medicine

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Variable analysis

independent variables

Anti-PRMT7 antibodies from Epicypher, Santa Cruz Biotechnology, and Millipore
Anti-Nanog, anti-Sox2, and anti-H4R3me2s antibodies from Active Motif
Anti-Oct4, anti-c-Myc, and anti-Klf4 antibodies from Cell Signaling
Anti-β-actin antibody from Sigma-Aldrich
Anti-H4R3me1 antibody from Thermo Scientific
Anti-H3 antibody from Abcam
Anti-mouse Argonaute 2 (Ago2) antibody from Wako Chemicals and Cell Signaling
CDNA constructs of human and mouse PRMT7 cloned into pCDH1-EF1-IRES-GFP vector
Human PRMT7's catalytic mutant (m. PRMT7) generated by mutating several residues

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Not specified

negative controls

Not specified

Annotations

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