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WBPicture0000012513 Description: Figure 5. Phosphomimetic NOS-3 Mutant Ser644Glu and Thr648Glu Reduces FBF Binding In Vitro and FEM-3 Accumulates during Oogenesis and Is Repressed in the Distal Germline by NOS-3 (A) Autoradiograph (top) showing binding analysis of recombinant Flag-tagged wild-type, S644/T648 sites mutated to alanine (phospho null) or S644/T648 sites mutated to glutamic acid (phosphomimetic) NOS-3 with S35-labeled FBF-1 (Supplemental Experimental Procedures). Wild-type and phospho null NOS-3 bind FBF-1 very robustly (lanes 2 and 3), while phosphomimetic NOS-3 results in a reduction of FBF-1 binding (lane 4) by 14-fold compared to wild-type. In replicate experiments, FBF-1 binding was reduced 17-fold or greater than 20-fold in two additional trials (data not shown). Photo (bottom) shows Coomassie blue staining of the input recombinant NOS-3 proteins used. (B and C) Adult hermaphrodite gonads stained for FEM-3 (green) and dpMPK-1 (red). (B) In wild-type, FEM-3, which is largely cytoplasmic, accumulates from mid-pachytene through the end of oogenesis mimicking that of dpMPK-1. FEM-3 is not detected in the distal germline. (C) In nos-3(oz231), FEM-3 accumulates throughout the germline, consistent with NOS-3 functioning in translational repression of fem-3 in the distal germline of wild-type. Dashed line marks the region of fem-3 translation repression by NOS-3.
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  Crop: Crop_picture: WBPicture0000012514
  Acknowledgment: Template: Reprinted from <Journal_URL>, <Article_URL>, Copyright <Publication_year>, with permission from <Publisher_URL>.
    Publication_year: 2011
    Article_URL: DOI id 10.1016/j.devcel.2011.04.009
    Journal_URL: DevelopmentalCell
    Publisher_URL: Elsevier
  Reference: WBPaper00038448