Activation of transcription factors by TLR ligands and antigen. IgE-primed MC/9 cells were stimulated or not (NS) for the periods indicated with 100 ng/mL LPS, 1 μg/mL P3C, and 20 ng/mL antigen (Ag), individually or in combination. Immunoblots were prepared from cell extracts and then probed for the indicated transcription factors or their activated phosphorylated forms with appropriate antibodies. Typical blots and their relative densities (mean ± SEM of values from 3 experiments) are shown for phosphorylated (Thr71)–ATF-2 (A), c-Jun (C), phosphorylated (Ser63)–c-Jun (D), and c-Fos (E). In addition, nuclear extracts were assayed for NF-κB (B) and c-Fos (F) oligonucleotide-binding activities by use of a commercial kit. The values are the mean ± SEM from 3 cultures. Identical results were obtained in a second experiment.