Nf κb2 p100 p52

Cells were lysed in phosphate-buffered saline (PBS) that contains 30 mM tris-HCl (pH 7.5), 120 mM NaCl, 2 mM KCl, 1% Triton X-100, and 2 mM EDTA supplemented with a protease inhibitor (Roche). Proteins were separated by electrophoresis on 4 to 15% precast polyacrylamide gels (Bio-Rad) and were transferred to 0.45 μm of nitrocellulose membrane (Bio-Rad). Membranes were blocked in a 1× solution tris-buffered saline/Tween 20 (TBS-T) with 5% nonfat dry milk for 1 hour at room temperature and then incubated overnight at 4°C with the specified primary antibody. Antigen-antibody complexes were visualized with peroxidase-conjugated secondary antibodies (GE Healthcare) and enhanced chemiluminescence Western blotting substrate (Pierce). Densitometry of immunoblots was done using the ImageJ analyzer software (1.48v).
Primary antibodies used in immunoblots were as follows: rabbit mAbs to β-actin (Cell Signaling Technology, 4970), HA tag (Cell Signaling Technology, 3724), IKKα (Cell Signaling Technology, 2682), NF-κB2 (p100/p52), (Cell Signaling Technology, 4882), NIK (Cell Signaling Technology, 4994), α-tubulin (Cell Signaling Technology, 2144), Lamin A/C (Cell Signaling Technology, 2032), SMAD2/3 (Cell Signaling Technology, 8685), and mouse mAb to Myc (Cell Signaling Technology, 2276). All secondary antibodies were obtained from the Jackson ImmunoResearch Laboratory.