Fig. 2. PGL-1 is retained by m⁷GTP-Sepharose through interaction with IFE-1. (A) Retention of native PGL-1 on m⁷GTP-Sepharose. Cleared lysates were prepared from wild-type (N2) worms and incubated with m⁷GTP-Sepharose. Bound proteins were eluted with m⁷GTP as described in Materials and Methods. To verify the specificity of cap interaction, either 100 µM GTP (G), 100 µM m⁷GTP (m⁷G) or buffer A (-) were added to lysates as competitors before chromatography. Eluted fractions (bound; 0.3 µg protein), column flow-through (unbound, 10% volume; 24 µg protein) and original lysate (lysate, 10% volume; 30 µg protein) were resolved by SDS-PAGE on a 6% gel and immunoblotted using anti-PGL-1 antiserum or anti-chicken actin antiserum (Sigma). (B) Retention of [³⁵S]PGL-1 on m⁷GTP-Sepharose via [³⁵S]IFE-1. [³⁵S]PGL-1 synthesized in reticulocyte lysate (total, t) is not retained on m⁷GTP-Sepharose (bound, b) unless IFE-1 is also synthesized in the lysate. Instead, PGL-1 alone is found entirely in the column flow-through (unbound, u). Equivalent volumes of total, bound and unbound fractions were resolved by SDS-PAGE on a 10% gel and ³⁵S-labeled proteins visualized by a phosphorimager. The asterisk indicates the migration of a protein of unknown identify (as seen in Fig. 1). (C) Depletion of IFE-1 in ife-1(RNAi) worms (generated by feeding; see Materials and Methods). m⁷GTP-binding proteins were enriched from wild-type (N2) or ife-1(RNAi) worm extracts, and analyzed by western blot (1.4 µg protein per lane) in order to detect endogenous IFE-1 and cap-retained PGL-1. Silver staining of a similar gel verified equal loading of m⁷GTP-binding proteins. Western blotting of total protein (30 µg per lane) from worm extracts verified the presence of PGL-1 and another eIF4E isoform, IFE-2, in ife-1(RNAi) worms.