Stimulation of mast cells by FcϵRI aggregation induces Egr-1 but not Sp-1 activation. (A) Mouse BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA (10 ng/mL) for various times. Nuclear proteins were isolated and subjected to EMSA analysis (see “Materials and methods”) using ³²P-labeled Egr-1– or Sp-1–specific probes. BLK indicates blank, no nuclear proteins were added; NT, no treatment, nuclear proteins were isolated from BMMCs without TNP stimulation. Numbers indicated are minutes after TNP stimulation; nuclear proteins were isolated from BMMCs after the indicated times following TNP stimulation. Fifty times concentrated unlabeled Egr-1 oligonucleotide was used to compete with ³²P-labeled Egr-1 oligonucleotide, whereas 50 × concentrated unlabeled AP-1 oligonucleotide was used as a nonspecific control probe. (B) Nuclear proteins from TNP-BSA (10 ng/mL, 60 minutes) treated BMMCs (TNP) or from untreated BMMCs (NT) were subjected to DNA probe competition experiment using unlabeled probes or ³²P-labeled mutant probes to demonstrate specific Egr-1 binding. No Sp-1 binding was observed in TNP-stimulated BMMCs. (C) As a control, nuclear extracts from HeLa cells (Promega) were subjected to EMSA using ³²P-labeled Sp-1 oligonucleotide. ³²P-labeled Sp-1 binding was blocked by unlabeled Sp-1 probe, but not by unlabeled Egr-1 or AP-1 probes (50 ×). (D) Antibody blockade of the DNA-protein complex formation. Nuclear proteins from TNP-BSA (10 ng/mL, 60 minutes) treated BMMCs or from untreated BMMCs (NT) were incubated with or without specific antibodies to Egr-1, Egr-2, Egr-3, or Egr-4 for 2 hours on ice before EMSA experiment using ³²P-labeled Egr-1 oligonucleotide.