Figure 1

(A) The purified proteins (1 or 2 µg) were subjected to SDS-PAGE under reducing condition and stained with silver or CBB, respectively. Two bands appeared in all the proteins (lanes 1–4). Molecular masses of the untagged esRAGE (lane 1) were approximately 49 and 52 kDa, while those of D₆-esRAGE, D₁₀-esRAGE and D₁₄-esRAGE were larger (lanes 2–4). For digestion of N-linked oligosaccharides, 2 µg each of the untagged and tagged proteins was denatured in denaturing buffer (0.5% SDS and 40 mmol/L dithiothreitol) at 100°C for 3 min, and the denatured proteins were then treated with 500 units of PNGase F in reaction buffer (50 mmol/L sodium phosphate [pH 7.5] and 1% NP-40) at 37°C for 1 h. The PNGase F-treated proteins were analyzed by SDS-PAGE after CBB staining. The PNGase F-treated protein showed a single band and smaller molecular mass than the untreated protein (lanes 5–8). (B) The purified proteins were mixed with HA suspension at a final concentration of 10, 20 and 40 Lig/mL. The unbound protein was separated from the bound protein by centrifugation, and the concentration of unbound protein in the supernatant was measured by Western blot analysis. The amount of bound protein was calculated by subtracting the unbound from the total. The longer acidic oligopeptide tagging showed the higher binding affinity of esRAGE to HA.