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.