Fig. 2. S100A13 is involved in the release of IL-1. (A) (left panel) The interaction of recombinant human IL-1 with S100A13 was assessed by the incubation of these proteins in 100 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl, followed by ultracentrifugation and S100A13 immunoblot analysis of the pellet fractions; (right panel) the Cu²⁺-dependent interaction of S100A13 with itself was assessed using the ultracentrifugation method described in the left panel. (B) Myc-S100A13 and IL-1-ßGal, insert-less vector and IL-1-ßGal NIH 3T3 cell cotransfectants were subjected to heat shock. Conditioned media were collected and processed as described (LaVallee et al., 1998). IL-1 was mmunoprecipitated with an IL-1 antibody, the immunoprecipitants were resolved by 8% and 12% acrylamide SDS-PAGE, respectively, and evaluated by IL-1 (top panel) and Myc (bottom panel) immunoblot analysis. (C) IL-1 NIH 3T3 cell transfectants were transiently transduced with a Myc-S100A13 adenoviral construct. 48 hours following transduction the cells were subjected to heat shock (42°C, 2 hours), cell lysates were obtained, immunoprecipitated with an anti-Myc antibody and resolved by IL-1 immunoblot analysis. (D) Myc-S100A13 and IL-1-ßGal NIH 3T3 cell cotransfectants and insert-less vector and IL-1-ßGal NIH 3T3 cell transfectants were evaluated for the release of IL-1-ßGal in the presence and absence of actinomycin D (10 µg/ml), as indicated, in response to heat shock. Conditioned media were processed and evaluated for IL-1-ßGal immunoblot analysis as described (Tarantini et al., 2001).