Fig. 4. Deletion of the basic residue-rich C-terminus functions as a dominant negative effector of IL-1 Release. (A) The recombinant form of S100A13 lacking the BR domain (S100A13BR) was incubated with recombinant IL-1 as described in Fig. 2A at the molar ratios indicated. (B) S100A13BR mutant and IL-1-ßGal NIH 3T3 cell cotransfectants were subjected to heat shock and, following DTT treatment, conditioned media were concentrated and immunoprecipitated with anti-IL-1 antibody for the evaluation of IL-1 release. Immunoprecipitated proteins were resolved by 12% (w/v) SDS-PAGE, and evaluated by IL-1 immunoblot analysis. (C) S100A13BR mutant and IL-1 NIH 3T3 cell cotransfectants as well as insert-less vector and IL-1 NIH 3T3 cell cotransfectants were transiently transduced with a wild-type S100A13 adenoviral construct; 48 hours following transduction the cells were subjected to heat shock and, following DTT treatment, media conditioned at either 37°C or 42°C for 2 hours was resolved by IL-1 immunoprecipitation followed by Myc immunoblot analysis (12% acrylamide SDS-PAGE).

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