Differences in NF-κB binding induction by IL-9 and TNF. (A) Delayed induction by IL-9 of a fast-migrating NF-κB complex. BW5147 cells were stimulated in the absence or in the presence of IL-9 (100 U/mL) or TNF (10 ng/mL) for 5 minutes or 24 hours. Nuclear extracts were prepared and a mobility shift assay was performed using a³²P-labeled palindromic κB site. The two types of NF-κB dimers binding to the probe (p65/p50 and p50 homodimers) are indicated. (B) NF-κB complexes induced by IL-9 contain only p50 subunits. BW5147 cells were stimulated in the absence or in the presence of IL-9 (100 U/mL) or TNF (10 ng/mL) for 24 hours. Nuclear extracts were prepared and a mobility shift assay was performed using a³²P-labeled palindromic κB site in the presence of anti-p50 (4 μg per lane) or anti-p65 (2 μg per lane) antibodies. The two types of NF-κB dimers binding to the probe (p65/p50 and p50 homodimers) are indicated. (C) IL-9 does not induce the phosphorylation and degradation of IκB. BW5147 cells were stimulated with 100 U/mL IL-9 or 10 ng/mL TNF for 5, 10, or 30 minutes or were left unstimulated. Total cell extracts were loaded on a gel and Western blotted. The membrane was hybridized with two antibodies: one directed against total IκB and another specific for phosphorylated IκB (Ser32).