The site-specific mutations, mSpi-1 and mNF-IL6 (Fig 2A) within the IL1B promoter block binding of Spi-1 and NF-IL6, respectively. DNA binding reactions were performed by using radiolabeled DT, DTmSpi-1, 6/IL-1, and 6/IL-1mNF-IL6 probes. DTmSpi-1 fragment containing a site-specific mutation, mSpi-1 (Fig 2A) was generated by restriction endonuclease digestion of 3MEHTmSpi-1. 6/IL-1mNF–IL6 is a synthetic oligonucleotide identical to the 6/IL-1, but possesses a site-specific mutation, mNF-IL6 described in the legend to Fig 2A. These mutations abolished IL1B promoter induction by Tax. Recombinant Spi-1 protein was incubated with either the wild-type DT (lane 1) or DTmSpi-1 (lane 2). Recombinant full-length NF-IL6 was incubated with either the wild-type 6/IL-1(lane 5) or its mutated version, 6/IL-1mNF-IL6 (lane 6). Nuclear extract prepared from THP-1 cells carrying pcTax, a Tax-expression vector was used in lanes 3, 4, 7, and 8.