Mature NcPI-S_3 ookinetes contain fewer micronemes.

<p>(A) Selected longitudinal sections, across the apical part of wild type (left image) and <i>NcPIS_3</i> mature ookinetes (middle and right images). All are surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white arrows), is a common feature in <i>NcPIS_3</i> ookinetes. The ookinete-specific characteristic structure of crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins CTRP and SOAP in <i>NcPIS_3</i> and <i>GFPp</i> ookinetes. No alteration was observed in transcript levels. <i>p28</i> was used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete extracts derived from <i>in vitro</i> cultured <i>NcPIS_3</i>, <i>NcPISmut</i> and <i>GFPp</i> ookinetes, with antibodies against the micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the <i>NcPIS_3</i> line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from <i>GFPp</i>, <i>NcPIS_3</i> and <i>NcPISmut</i> ookinete cultures were probed with antibodies against phosphorylated eIF2α. <i>NcPIS_3</i> parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker, BiP was used as the loading control.