Fig. 4.

Depletion of P1 and P2 proteins from rat ribosomes and its effect on ribosome binding by RTA. (A) Ribosomes were treated with NH₄Cl (0.4M) followed by the addition of ethanol (50% [v/v]) to selectively remove P1and P2. Following high speed centrifugation, the ribosomal pellet and the supernatant containing stripped P1/P2 proteins were collected and analyzed by western blotting with a monoclonal antibody against the conserved C-termini of P-proteins. The indicated P1/P2 protein levels in depleted ribosomes are expressed as a percentage the P1/P2 protein levels in untreated samples, normalized to P0 levels. (B) The interaction of RTA with WT and P1/P2-depleted rat ribosomes measured by surface plasmon resonance. N-terminal His-tagged RTA was coupled to an NTA chip on the Biacore 3000 and ribosomes (5 nM) were passed over the surface for 5 min and dissociation was monitored for 10 min. The interaction of ribosomes with a control ligand (EGFP) was analyzed in parallel and subtracted from the signal obtained for the target surface (RTA), to account for non-specific binding.