Constitutive NF-κB activation and bcl-2 expression in tal-1 and muttal-1 tumors. (A) Nuclear extracts were prepared from wild-type thymocytes, tal-1 tumors, and mut tal-1 tumors and incubated with a radiolabeled NF-κB consensus oligonucleotide probe. As a positive control for NF-κB activation, nuclear extracts were also prepared from murine embryonic fibroblasts (mef) left untreated or treated with the cytokine TNF-α. The binding reactions were fractionated on a 5% polyacrylamide gel and the DNA-protein complexes were detected by autoradiography. (B)NF-κB complex in tal-1/scl tumors contains p65/p50. Binding reactions were incubated with antibodies against p50, p65, and c-rel prior to separation on a nondenaturing gel. (C) Total cell lysates (50 μg) were fractionated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and immunoblotted with an anti-bcl-2 antibody. Lysates were probed with an anti-β-actin antibody to control for the amount of total protein.