Tethered NOS-3 increases expression of pgl-1 endogenous gene. (A) Tethering assay using SNAP-tagged pgl-1 gene as the reporter. Endogenous pgl-1 includes a SNAP tag to visualize its protein product and 3× boxB hairpins to recruit NOS-3:: λN::FLAG to its 3′UTR. (B) Tethered NOS-3 increases expression of PGL-1 protein in the worm germline. Confocal images (max projection) of adult gonads stained for PGL-1::SNAP (SNAP ligand), NOS-3 (αFLAG), and DNA (DAPI). Wild-type worms serve as a negative control. (C) Quantitation of PGL-1::SNAP. SNAP ligand signal was averaged across the distal 5–105 µm of germlines. Wild-type (N2, no PGL-1::SNAP reporter; n = 44), NOS-3::FLAG (n = 33), NOS-3:: λN::FLAG (n = 41) worms analyzed. (***) P < 0.0001. (D) Immunoblot to assay PGL-1 (αSNAP) and NOS-3 (αFLAG) protein expression. Actin serves as the loading control, and wild-type worms as a negative control (first lane). Whole worm lysate (n = 30 young adult worms) used for all samples.
