Figure 4From: A novel receptor – ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor TuIdentification of bacterial ligands for nucleolin. Part A. Samples were prepared as outlined in Experimental Procedures, normalized to 50 μg proteins per lane before separation by SDS-PAGE. Immunoblotting (I.B.) was performed either with anti-EF-Tu Ab diluted 1/100,000 (part A1) or anti-IglA Ab diluted 1/5,000 (part A2). Part B. 500 μg LVS membrane proteins were incubated with different concentrations of biotinylated p63 peptide (p63*). Complexes, formed between bacteria proteins and biotinylated p63 peptide, were isolated by purification using avidin-agarose. Purified proteins were analyzed by SDS-PAGE. Immunoblotting (I.B.) was performed with anti-EF-Tu Ab diluted 1/100,000. Mobility of marker proteins is indicated on left side. Part C. Specific binding of EF-Tu present in LVS membranes with RGG domain of nucleolin. 500 μg LVS membrane proteins were preincubated without (lane -) or with 50 μM unlabeled p63 peptide, before incubation with 5 μM biotinylated p63 peptide (p63*). Complex formed between EF-Tu present in bacteria membrane proteins and biotinylated p63 peptide was analyzed by immunoblotting with anti-EF-Tu Ab diluted 1/100,000. In control, 50 μg LVS membrane proteins were run directly.Back to article page