Fujitani K , Otomo A , Nagayama Y , Tachibana T , Kato R , Kawashima Y , Kodera Y , Kato T , Takada S , Tamura K , Takamatsu N , Ito M .

	Figure 1. Immunoreaction of an anti-DMRT1 monoclonal antibody 4F6 against X. laevis DMRT1. (A) Immunoblot analysis using the anti-FLAG (M5) or anti-DMRT1 (4F6) monoclonal antibodies. pcDNA3-FLAG or pcDNA3-FLAG-DMRT1 was transiently transfected into 293T cells. Extracts of 293T cells were examined by immunoblotting with each antibody followed by a HRP conjugated anti-mouse IgG antibody. DMRT1 was detected as a single band at the same size by both antibodies (arrowhead). (B) Immunoprecipitation (IP) analysis with the anti-FLAG (M5) or anti-DMRT1 (4F6) monoclonal antibodies. pcDNA3-FLAG-DMRT1 was transiently transfected into 293T cells. The cell lysate was mixed with each antibody and pulled down with protein A/G agarose. IP extracts were examined by immunoblotting using the anti-DMRT1 polyclonal antibody followed by a HRP conjugated anti-rabbit IgG antibody. (C) Immunohistochemical analysis using the anti-DMRT1 monoclonal antibody 4F6 (1/10) and anti-DMRT1 polyclonal antibodies (1/1000) . Frozen sections of adult testis were stained with Hoechst 33258 for nuclei and reacted with both the antibodies, followed by Alexa 594-conjugated anti-mouse (red) and Alexa 488 anti-rabbit-IgG (green) antibodies. Both signals showed the same staining patterns.
	Figure 2. Silver staining of immunoprecipitates using the anti-DMRT1 monoclonal antibody 4F6 from X. laevis adult testes. Testis extracts were mixed with normal mouse IgG or the anti-DMRT1 antibody 4F6, and pulled down with protein A/G agarose. The immunoprecipitates were examined by silver staining (the right and middle lanes) or immunoblotted with an anti-DMRT1 polyclonal antibody (right lane). Seven 4F6-specific bands were excised, and examined by LC-MS.
	Figure 3. Effects of PACT/PRKRA and/or p53 on transcriptional activity by DMRT1 using luciferase reporter assay. 150 ng of DMRT1-driven firefly luciferase reporter plasmid (p4xDMRT1-luc), 3.3 ng of DMRT1 expression plasmid (pcDNA3-FLAG-DMRT1), and 10 ng Renilla luciferase vector (pRL-TK-luc) as transfection internal control in the presence or absence of PACT/PRKRA and/or p53 expression plasmids (pcDNA3-FLAG-PACT/PRKRA and/or -p53) were transiently co-transfected into 293T cells, using 1.2 μg PEI MAX. Total amount of DNA was kept at 250 ng per each transfection with pcDNA3-FLAG empty vector. 24 hours after transfection, cell lysates were used to measure luciferase activity. Relative activity is shown as the fold increase compared with the value obtained with 250 ng of pcDNA3-FLAG empty vector. The symbols -, +, and ++ indicate 0, 3.3, and 20 ng, respectively. Values are expressed as mean ± SE, n = 3. The letters above the bars indicate the results of Tukey HSD test following one-way ANOVA (p < 0.05).
	Figure 4. Expression of PACT/PRKRA mRNA in developing ZW and ZZ gonads. (A) Quantitative RT-PCR analysis of Pact/Prkra mRNA during gonadal development of ZW (red) and ZZ (blue) tadpoles and adults in X. laevis. cDNAs were synthesized using total RNAs from ZW and ZZ gonads at various stages of tadpoles after sex determination, and at 6 weeks, 1 year, and 2 years of frogs after metamorphosis, and then amplified by PCR using specific primer pairs as described in Table S1. W and Y show weeks and year(s), respectively. EF1α was used for normalization. RT-qPCR data represent the mean (n=3) and SD. Values are expressed as mean ± SE, n = 3. Differences among stages were evaluated by one-way ANOVA followed by the Tukey HSD test (p < 0.05). Mean values not sharing the same letters are significantly different from each other. Sexual differences between ZZ and ZW gonads at each stage were evaluated or by Student’s t-test (*p < 0.05). N.S., not significant. (B) Distribution of Pact/Prkra mRNAs on transverse sections of ZW and ZZ tadpole gonads at stage 56. Whole-mount in situ hybridization of the gonads with the attached mesonephros was performed with the Pact/Prkra sense or anti-sense RNA probe, followed by 7-μm cryostat sectioning. The sections were treated with an anti-VASA monoclonal antibody to identify germ cells (red) and Hoechst 33258 for nuclei (blue). Note that the nuclei in germ cells were faintly stained by Hoechst 33258. Arrowheads indicate Pact/Prkra-expressing germ cells. Scale bars, 20 μm.

Bavelloni, Prohibitin 2: At a communications crossroads. 2015, Pubmed

Bavelloni, Prohibitin 2: At a communications crossroads. 2015, Pubmed
Bennett, The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G₁ arrest. 2012, Pubmed
Cuddihy, The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro. 1999, Pubmed
Fujitani, Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis. 2016, Pubmed , Xenbase
Gebel, Control mechanisms in germ cells mediated by p53 family proteins. 2017, Pubmed
Huang, The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR). 2002, Pubmed
Ito, JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway. 1999, Pubmed
Krentz, DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary. 2011, Pubmed
Li, PACT is a negative regulator of p53 and essential for cell growth and embryonic development. 2007, Pubmed
Masuyama, Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka. 2012, Pubmed
Matson, DMRT1 prevents female reprogramming in the postnatal mammalian testis. 2011, Pubmed
Matson, The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. 2010, Pubmed
Matsuda, DMY is a Y-specific DM-domain gene required for male development in the medaka fish. 2002, Pubmed
Mawaribuchi, Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis. 2017, Pubmed , Xenbase
Mawaribuchi, Molecular Evolution of Two Distinct dmrt1 Promoters for Germ and Somatic Cells in Vertebrate Gonads. 2017, Pubmed , Xenbase
Mawaribuchi, Molecular evolution of vertebrate sex-determining genes. 2012, Pubmed , Xenbase
Nanda, A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. 2002, Pubmed
Okamoto, Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression. 2000, Pubmed
Patel, PACT, a protein activator of the interferon-induced protein kinase, PKR. 1998, Pubmed
Redfern, RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators. 2013, Pubmed
Sado, Lymphocytes from enlarged iliac lymph nodes as fusion partners for the production of monoclonal antibodies after a single tail base immunization attempt. 2006, Pubmed
Smith, The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. 2009, Pubmed
Takashima, Regulation of pluripotency in male germline stem cells by Dmrt1. 2013, Pubmed
Wada, Masculinization-Related Genes and Cell-Mass Structures During Early Gonadal Differentiation in the African Clawed Frog Xenopus laevis. 2017, Pubmed , Xenbase
Yoshimoto, Expression and promoter analysis of Xenopus DMRT1 and functional characterization of the transactivation property of its protein. 2006, Pubmed , Xenbase
Yoshimoto, Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system. 2010, Pubmed , Xenbase
Yoshimoto, A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis. 2008, Pubmed , Xenbase
Yoshimoto, A ZZ/ZW-type sex determination in Xenopus laevis. 2011, Pubmed , Xenbase
Zhao, Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1. 2015, Pubmed