Figure 5

Recombinant EF-Tu specifically interacts with RGG carboxy-terminal domain of nucleolin. A. P63 peptide, corresponding to RGG domain of nucleolin binds in dose-dependent manner to recombinant EF-Tu. Aliquots containing 1 μg recombinant His-EF-Tu were incubated with different concentrations of biotin-labeled p63 peptide. Complex formed between His-EF-Tu and biotin-labeled p63 peptide was isolated by purification using avidin-agarose. Purified proteins were analyzed by SDS-PAGE. Lane S shows purified preparation of 0.2 μg His-EF-Tu. Presence of EF-Tu was detected by immunoblotting using anti-EF-Tu Ab diluted 1/100,000. B. Specific binding of recombinant EF-Tu with RGG domain of nucleolin. Aliquots containing 1 μg of His-EF-Tu were preincubated without (lane -) or with 40 or 80 μM unlabeled p63 peptide before further incubation with 4 μM biotin-labeled p63 peptide. Biotin-labeled p63 peptide (4 μM) was also preincubated with HB-19 (40 or 80 μM), 80 μM F3 or 80 μM 9Arg (9R) peptides before further incubation with 1 μg His-EF-Tu. Complexes formed between His-EF-Tu and biotin-labeled p63 peptide were isolated and analyzed by immunoblotting as in section A. On left is position of molecular weight protein markers. On right is position of 43 kDa His-EF-Tu. C: 5 μg GST (lane 2) or GST-EF-Tu (lane 3) bound on glutathione-Sepharose beads were incubated with THP-1 solubilized membrane proteins (500 μg). After extensive washes, bound proteins were run on SDS-PAGE with, in control, 50 μg proteins solubilized from THP-1 membranes, run directly without incubation with beads (lane 1). Immunoblotting was performed with anti-nucleolin MAb, diluted 1/10,000. Estimated molecular mass (in kDa) of nucleolin is indicated on left of black line.