GLS-1 and Germline Development Figure 3. GLS-1 Binds GLD-3 Specifically. (A) Protein interactions as assayed in the yeast 2-hybrid system. b-galactosidase activities of yeast cells co-expressing protein pairs fused to the LexA DNA binding domain (DB) and the Gal4 transcriptional activation domain (AD) are given in arbitrary units. (B) Binding of GLS-1, GFP and GLD-3 co-produced in insect cells. Protein pulldowns with either MBP-GLD-3L or bacterially expressed MBP. Extracts were either RNAse A or mock incubated to demonstrate a non-RNA mediated interaction (bottom panel). The effectiveness of RNA digestion was monitored on an agarose gel (right panel). (C) Whole worm extracts were incubated with anti-GLS-1, anti-GLD-3L immune sera (IS) or the corresponding pre-immune serum (Pre-IS). Precipitated material was detected with the reciprocal antibodies. A prior treatment of the extract with (+) or without (2) RNase A treatment was monitored for efficiency on an agarose gel in the case of the GLD-3-coIP (not shown). (D) Identification of the minimal GLS-1 binding domain in GLD-3L. The strength of interaction with GLS-1 is rated according to the blue color of a b-galactosidase substrate (+ and 2 represent strong and weak binding, respectively). FL, full-length. (E) Identification of the minimal GLD-3 binding domain in GLS-1. The strength of interaction with GLD-3L on a filter assay is given and labeled as in (D). doi:10.1371/journal.pgen.1000494.g003 The GLS-1 interaction with MBP::GLD-3L was still observed (Figure 3B). In addition, we confirmed the existence of an in vivo GLS-1/ GLD-3 complex by co-purifying each protein from wild-type worm extracts using protein-specific antibodies coupled to agarose beads (Figure 3C). Regardless of RNAse A treatment, GLD-3 and GLS-1 were strongly enriched in the anti-GLS-1 or anti-GLD-3 immunoprecipitate, respectively, but not in the IgG controls (Figure 3C). Finally, we used protein truncations to map the interaction domain between GLD-3 and GLS-1 in yeast 2-hybrid tests. We detected two interaction surfaces within GLD-3L; an N-terminal PLoS Genetics | www.plosgenetics.org 5 May 2009 | Volume 5 | Issue 5 | e1000494