Fig. 4. GLD-1 selectively binds pal-1 3' UTR elements that mediate germline repression in vivo. Biotinylated 3' UTR RNA fragments were incubated with extracts prepared from adults expressing a rescuing GLD-1::GFP::FLAG fusion protein (GGF). Biotinylated RNA was isolated using streptavidin magnetic beads and bound proteins were subjected to SDS-PAGE and western analysis with anti-FLAG antibody. S, sense RNA; AS, antisense RNA. This assay shows the expected specificity, as GGF is captured by tra-2 3' UTR RNA that bears GLD-1 binding sites, but not by a tra-2 antisense RNA. 2X GRE, A, and B RNAs capture GGF, while antisense 2X GRE and C RNAs do not, showing that binding activity in extracts correlates with in vivo repression activity.