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	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.

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