Electrophoretic mobility shift assays: Protein binding activity in nuclear extracts from untreated (co), TNFα- (T), PTX- (X), and TNFα/PTX (TX)-treated HL-60 cells to the 18-bp (A) or the 19-bp (B and C) sequence motif. Specificity of the DNA-protein interaction was determined by competition experiments (A and B) using unlabeled oligonucleotides containing the 18-bp (18), the 19-bp (19), or the 21-bp (21) sequence motifs of the IE enhancer or an oligonucleotide containing the CRE consensus sequence (CRE). For competition nuclear extracts were preincubated for 5 minutes with a 30 to 50 molar excess of unlabeled oligonucleotides before addition of the radiolabeled probe. The protein complex bound to the 19-bp sequence motif was characterized by antisera reactive to CREB-1, ATF-1, c-jun, or CREM-1 (C). The reaction mixture was supplemented with 1.5 μL antisera. Arrows on the right point to a new, highly retarded protein-DNA-antibody complex. The autoradiograph shows one representative experiment that was repeated at least three times.