Immunoblotting and EMSA to Characterize NF-κB Pathway

Immunoblotting was performed using whole cell lysate as previously described^{37 (link)}. Primary antibodies used include: anti-p50 (sc7178, Santa Cruz Biotechnology), anti-GAPDH (sc-137179,), anti-CDK1 (sc-54), anti-HA (sc-7392), anti-actin (sc-1616). Unedited, full-length immunoblots are provided in Supplementary Information (Supplementary Figs. S7–Fig. S15). Several immunoblots were cut prior to antibody hybridization for reagent conservation. To identify the κB-site, the program TFSEARCH was used and a sequence with 86% homology with the canonical κB binding site was identified. For EMSA, nuclear fraction was isolated, a double-stranded oligonucleotide (5′-CGCTGAAGAGAATTCCCAAGGC-3′) (IDT) containing the decameric κB-consensus sequence was end labeled with [ɣ-³²P] ATP and used as a probe, and the assay performed as previously described^{13 (link)}. Supershift assays were performed using antibodies specific to p50 or BCL-3. Competition was performed by pre-incubating the mixture with cold specific and non-specific DNA probe.