<p>(<b>A–F</b>) Larvae were subjected to <i>pbs-3</i>(<i>RNAi</i>) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1, L2 and L3 stages, respectively. (A), (C) and (E) show anti-SPD-2 alone. (<b>G–H</b>) Larvae were subjected to <i>pbs-3</i>(<i>RNAi</i>); <i>spd-2</i>(<i>RNAi</i>) and subsequently stained with DAPI (red) and anti-SPD-2 (green) in the L1. (G) shows anti-SPD-2 alone. Asterisks indicate the intestinal nuclei. Scale bar, 5 µm. (<b>I</b>) SPD-2 nuclear localization was monitored and the number of intestinal cells that demonstrate diffuse nuclear SPD-2 staining was compared in control and <i>pbs-3</i>(<i>RNAi</i>). (<b>J</b>) The effects of <i>pbs-3</i>(<i>RNAi</i>) on SPD-2 stability were quantified by determining the number of intestinal cells that express SPD-2 at later larval stages. Error bar, standard deviation; n≥50; P<0.05 (t-test). (<b>K–M</b>) SAS-4 levels were stained in the intestinal cells of late L2 <i>pbs-3</i>(<i>RNAi</i>) animals and were quantified as above (<b>N</b>). The asterisks indicate the intestinal nuclei, while arrowheads point to SAS-4 foci. Scale bar, 5 µm. n≥50; P<0.05 (t-test). (<b>O</b>) Homogenates obtained from animals expressing 3XFLAG tagged SPD-2 subjected to <i>pbs-3</i>(<i>RNAi</i>) or <i>gfp</i>(<i>RNAi</i>) (control), were incubated with anti-FLAG antisera and associated proteins were immunoprecipitated with Protein A-agarose. The pellets were blotted with anti-ubiquitin or anti-SPD-2 respectively. kDa, kilo Dalton.</p
