Monocyte transendothelial migration is inhibited by anti-amphoterin and anti-RAGE antibodies, and by soluble fragments of RAGE. (A) Western blotting of monocyte and HUVEC lysates with the anti-Atn used in the migration assay. Cells were lysed in hot reducing Laemmli sample buffer, and the lysates were analyzed in Western blotting on 10% to 20% gradient gel. recAtn was used as a standard. Western blotting experiments revealed specifically a 30-kD band. (B) Transendothelial migration assay of monocytes. Monocytes were added to upper chambers of HUVEC-coated Transwell filters. Monocytes were allowed to migrate for 3 hours. Various concentrations of antibodies were added to the upper well at the start of the experiment where indicated. Migrated monocytes were counted microscopically. Noninhibited migration was defined as 100%. Bars represent ± SD (n = 3). (C) Monocyte transendothelial migration is inhibited by sRAGE isolated from bovine lung. Monocytes were added to the upper wells of the Transwell chambers with various concentrations of sRAGE. Noninhibited migration was defined as 100%. Migration assay was done as in Figure 5B. (± SD, n = 3). (D) Monocyte transendothelial migration is inhibited by recombinant sRAGE-Ig and the RAGE V₁ domain. Monocytes were added to the upper wells of the Transwell chambers with 40 g/mL sRAGE-Ig, V₁-Ig, or Fc-control (Ig). Noninhibited migration was defined as 100%. Migration assay was done as in Figure 5B (± SD, n = 8 for noninhibited, Ig, and sRAGE-Ig wells, and n=4 for V₁-Ig wells). (E) Various concentrations of anti-RAGE antibodies (anti-P300 and anti-P301) were added to upper well at the start of the experiment where indicated. Nonimmune IgG was used as a control. Migration assay was done as in Figure 5B. Noninhibited migration was defined as 100% (± SD, n = 4, ^*P < .00005).