Fig. 7. A box competes with HMGB1 for cell surface binding. (A) ¹²⁵I-HMGB1 binding was measured in RAW 264.7 cells plated in 24-well dishes for the times indicated. Specific binding was determined as total binding minus the radioactivity obtained in the presence of 5,000-fold molar excess of unlabeled HMGB1 [total cell-associated ¹²⁵I-HMGB1 (cpm per well) minus cell-associated cpm per well in the presence of 5,000-fold molar excess of unlabeled HMGB1]. (B) "Total" equals cpm per well of cell-associated ¹²⁵I-HMGB1 in the absence of unlabeled HMGB1 or A box for 2 h at 4ºC. "HMGB1" or "A box" equals cpm per well of cell-associated ¹²⁵I-HMGB1 in the presence of 5,000-fold molar excess of unlabeled HMGB1 or A box. Data (from three to four separate experiments) are expressed as percent of total cpm obtained in the absence of unlabeled HMGB1 proteins (2,382 ± 179 cpm per well).