Interaction of AAT-6 with NRFL-1 <i>in vivo</i>. <i>A</i>

<p>, Expression of AAT-6 in worms. AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> was localized to the luminal surface (<i>single arrowhead</i>) but not to the basal side (<i>double arrowhead</i>) of the intestinal epithelia. Scale bar, 25 µm. Non-specific fluorescence on gut granules was seen. More than twenty worms were analyzed. <b><i>B</i></b>, Co-localization of AAT-6 and NRFL-1. GFP fluorescence from AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> (<i>top</i>) was co-localized with immunostaining of NRFL-1 by anti-NRFL-1 antibody visualized by Cy3-labeled secondary antibody (<i>middle</i>). Bottom image is merged from top and middle images. Confocal images of a representative intestine section (whole worm) are shown. Scale bar: 25 µm. More than five worms were analyzed. <b><i>C</i></b>, Intensity profile along the line A–B in the merged image shows an overlapping of the peaks of the NRFL-1and the AAT-6 signal. <b><i>D</i></b>, Immunoprecipitation of NRFL-1/AAT-6 complex from worm lysate. The worm lysate was immunoprecipitated with anti-GFP monoclonal antibody (mouse) and immunoblotted using anti-NRFL-1 antibody. <i>Top</i>: input (2.5%). <i>Middle</i> and <i>bottom</i>: immunoprecipitant was immunoblotted using anti-NRFL-1 antibody and anti-GFP antibody (chicken), respectively. In the middle blot, two major bands of NRFL-1 detected (<i>arrowheads</i>), probably reflecting partial dephosphorylation during immunoprecipitation process. A representative blot of two separate experiments is shown.