Fig. 1. The stress-induced release of IL-1, FGF1 and S100A13 is Cu²⁺-dependant. (A) Recombinant human IL-1 (1 µg in 100 mM Tris HCl, pH 7.2) was adsorbed to a Cu²⁺-chelating column (Hi-Trap, Amersham), the column was washed with five column volumes of 0.2 M sodium phosphate buffer, pH 7.2 and eluted with an imidazole gradient in the wash buffer. The column was stripped with 50 mM EDTA and all fractions including the flow-through fraction were resolved by 15% acrylamide SDS-PAGE and evaluated using an IL-1 antibody. (B) Conditioned medium obtained from heat-shocked PMA-stimulated U937 and stable FGF1 U937 cell transfectants was processed by Cu²⁺ chelator affinity chromatography. Eluted proteins were resolved by 15% acrylamide SDS-PAGE, and evaluated by IL-1 (top-left panel), FGF1 (top-right panel) and S100A13 (bottom panel) immunoblot analysis. (C) NIH 3T3 cells stably transfected with IL-1 were either incubated for 18 hours at 37°C in the absence and presence of the Cu²⁺ chelator, TTM or for 2 hours in the absence or presence of the specific calpain inhibitor, ZLL, as indicated and the untreated and treated cells either maintained at 37°C or subjected to heat shock. The conditioned medium was processed and evaluated for IL-1 release as described in A. Cell lysates from TTM- and ZLL-treated cells were used to monitor the intracellular level of IL-1 expression. The TTM- and ZLL-negative control cell lysates exhibited a similar level of IL-1 expression (data not shown).