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Fig. 3. Biochemical support that the C-terminal residues of EBP50 bind as proposed. (A) Residues F355 and L358 in the EBP50 tail are important for the interaction with the FERM domain. Bacteria containing plasmids for the expression of MBP fused to the C-terminal 39 residues of EBP50 (WT), or containing the F355R mutation (F355R) or the last residue deleted (L358
), were grown to log phase and either subjected to induced protein expression with IPTG (+) or not induced (-). Lysates were prepared and applied to beads on which the ezrin FERM domain had been immobilized. After washing, bound proteins were eluted and analyzed. The three eluates show proteins recovered from the induced lysates; essentially no material was recovered from parallel uninduced lysates (not shown). (B) The C-terminal 14 residue peptide of EBP50 binds to the FERM domain. Purified wild-type ezrin FERM domain (Wild type) or purified ezrin mutant N210F/T214A FERM domain (N210F/T214A) was mixed with beads containing covalently linked peptide (Peptide) or beads lacking the peptide (Control). The load (L), unbound (U), and bound and eluted (B) fractions were analyzed by SDS-PAGE.
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