Western blotting analysis of amphoterin and RAGE. (A) Leukocytes were lysed with 1% SDS, and 10-g samples of total cellular protein or 40 ng recAtn were run under reducing conditions on SDS-PAGE and transferred to a nitrocellulose filter. The filter was immunostained with anti-recAtn antibodies. PMN indicates polymorphonuclear leukocytes. (B) Monocyte lysates (40 μg protein) and recAtn (80 ng) were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were immunostained with 5 affinity-purified antibodies raised against different amphoterin peptides (I-V). (C) Rat brain microglia lysates were analyzed in Western blotting with anti-recAtn and antipeptide II. Both antibodies recognized a single 30-kD band from the lysate. (D) sRAGE was purified from bovine lung acetone powder. Purified sRAGE migrated as a single band in 12% SDS-PAGE stained with Coomassie blue (lane i). sRAGE was detected by 4 different anti-RAGE antibodies in 10% to 20% SDS-PAGE and Western blotting experiment: anti-P300 (lane ii), anti-P301 (lane iii), anti-RAGE (lane iv), and anti-RAGE N-16 (lane v). (E) Western-blotting of monocyte RAGE. RAGE was detected from 1-hour amphoterin adherent human monocytes or rat leukocytes using anti-P300 (lane i), anti-RAGE (lane ii), or anti-P301 (lane iii). Lanes i-ii: human cells. Lane iii: rat cells.