Figure 2.

eIF4B binds to eIF4A in the presence of ADPNP and RNA. (A) Schematic overview of the supernatant depletion assay after Pollard et al.²² to probe binding of eIF4B to eIF4A. Increasing amounts of streptavidin beads with immobilized biotinylated eIF4A (eIF4A-bio, orange sphere) or without (negative control, red sphere) were incubated with a constant concentration of eIF4B or Alexa-488 labeled eIF4B (eIF4B-A488). The supernatant is analyzed by SDS-PAGE. A decrease of protein in the supernatant reflects binding to eIF4A-bio. (B) Supernatant depletion assay to follow binding of 0.25 µM eIF4B-A488 (eIF4B_248C/274C) to eIF4A-bio in the presence of 5 µM RNA (32mer) and 10 mM ADPNP (Coomassie Blue staining). eIF4A-bio concentrations are 0, 4, 8, 12, 15 and 33 µM. eIF4B in the supernatant was quantified from the fluorescence intensity. Red: depletion upon addition of streptavidin beads (negative control), yellow: depletion upon addition of eIF4A-conjugated streptavidin beads. Data shown are a representative example of two independent experiments (see Fig. 3C). (C), (D) Supernatant depletion assay with eIF4B in the absence of ADPNP or RNA.