Figure 3. Expression and protein profile of NRFL-1 (C01F6.6) in C. elegans. A-D, Expression of NRFL-1 in worm. gfp::nrfl-1
expression was detected in excretory canal (arrow in A and B) and luminal membrane of intestinal epithelial cells (single-arrowhead ) in the anterior (B), middle (C) and posterior (D) intestine. gfp::nrfl-1
was not detected on the basal side (double-arrowhead in B, C and D). In A, a worm with gfp::nrfl-1
expression restricted to the excretory system was imaged for clarity. Such worms occasionally occurred in the transgenic population. In C and D, cytosolic dispersion of NRFL-1 was seen. Scale bars, 25 mm. Ten worms examined for each. E, Luminal enrichment of NRFL-1. Endogenous NRFL-1 was detected by anti-NRFL-1 antibody along the luminal side of the intestine. Non-specific signal on the body-wall is arrowed (left). IFB-2 was immune-labeled in a similar pattern (middle). Merged image shows that NRFL-1 is distributed apical to IFB-2 (right). Scale bar, 25 mm. Confocal images of a representative intestinal section (whole-worm) from seven independent experiments are shown. F, Immunoblot of endogenous and recombinant NRFL-1. Left (recombinant), middle (wild type) and right (nrfl-1
) lanes are for recombinant C01F6.6a and C01F6.6b proteins (untagged), lysate from wild-type, and lysate from nrfl-1
), respectively. Note that the band at ,72 kDa in the middle lane was not detected in the right and the band did not match either C01F6.6a (,62 kDa) or C01F6.6b (,78 kDa). Sixty microgram of protein was loaded for the lysates. A representative blot from three separate experiments is shown. G, Dephosphorylation of endogenous NRFL-1. The wild-type lysate was incubated with (+) and without (2) phosphatase inhibitors for the indicated period. Over incubation, the bands for inhibitor-free lysate migrated towards a position corresponding to the recombinant C01F6.6a. Ninety microgram of protein was loaded for the lysate. The results are confirmed in duplicate experiments.