Maintenance of AAT-6 on the intestinal luminal membrane by NRFL-1. <i>A</i>

<p>, The localization of AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> was compared between <i>aat-6</i> and <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> genetic backgrounds. Epifluorescence images of the distribution of AAT-6 in the intestine are shown for worms two, four and six days after hatching. In six-day old worm, the membranous localization of AAT-6 decayed in <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i>, whereas AAT-6 was retained on the luminal membrane in six days in the presence of NRFL-1 (<i>aat-6</i>). Scale bars: 100 µm. Representative pictures from more than ten worms analyzed for each are shown. <b><i>B</i></b>, Fluorescence intensity was measured to quantify the age-dependent regulation. The intensity of the intestinal luminal surface was peaked at day four with significantly stronger signal in <i>aat-6</i> (gray column) compared with <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> (white column). The intensity at day six did not differ significantly between the strains (<i>luminal membrane</i>). <b><i>C</i></b>, The fluorescence intensity of the whole intestine showed a similar pattern (<i>whole intestine</i>). The localization index, luminal intensity divided by whole intestine intensity, quantifies the membranous localization. The six-day old <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> worm had a significantly lower score, showing age-dependent decay in luminal localization (<i>localization index</i>). Gray column, <i>aat-6</i>. White column, <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i>. Values are presented with mean ± S.E. (n = 5). <b><i>D</i></b>, Immunoblot and densitometric analysis of AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> in six-day old worm. Densitometric analysis followed by anti-GFP antibody immunobloting exhibited no significant difference in band intensity between the genetic backgrounds. A representative blot was presented with actin as a loading control. The bar graph indicates the relative band intensities of the respective sample. Values are presented with mean ± S.E. (n = 4).