Sc 7607

Wild type (WT) and Ikk2^−/− mouse embryonic fibroblast (MEFs) were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 2 mM Na pyruvate and 1x PSF (penicillin, streptomycin and fungizone) at 37 °C with 5% CO₂. Antibodies against IKKα (Sc7182, Sc7218), IKKβ (Sc7607), IKKγ (Sc8330) were from Santa Cruz Biotechnology and Biosource. The anti-IKKα/β rabbit IgG antibody (sc-7607, Santa Cruz Biotechnology) recognizes human IKK1 and IKK2 both. IKKγ specific antibody was obtained from Biosource and Millipore. TNFα (Calbiochem) were used at concentration of 10 ng/ml.

Tissue was snap-frozen in liquid nitrogen, pulverized, and resuspended in a buffer containing 4% SDS, 100 mM Tris-HCl, and protease/ phosphatase inhibitors (Roche). 10 to 20 μg of protein were separated on 4–12% gradient gels (Invitrogen). Proteins were transferred to a PVDF or nitrocellulose membrane with a semi-dry blotter (Bio-Rad). Membranes were blocked for 1 h (RT) in TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk, incubated with primary antibody over night (6°C) in TBS, 0.1% Tween-20 with 5% nonfat dry milk or 5% BSA, and then incubated with HRP-coupled secondary antibody for 1 h (RT). After application chemiluminescence reagent, membranes were exposed to x-ray films. Primary antibodies against the following antigens were used: human IKK2 (Abcam Y466), IKK1/2 (Santa Cruz sc-7607, lot H2208), ERK2 (Santa Cruz sc-154, lot F0210), p21 (Santa Cruz sc-6246, lot A1209), Stat1 (Cell Signaling 9172, lot 14), phospho-Stat1 (Cell Signaling 9167, lot 6), and luciferase (Promega G7451).

The following antibodies were used: antibody against NEMO (FL-419, sc-8330, Santa Cruz Biotechnology), Flag-HRP (A8592, Sigma), Tubulin (CP06, EMD Millipore), Actin (sc-1616, Santa Cruz Biotechnology), and IKKα/β (sc-7607, Santa Cruz Biotechnology). Anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase were obtained from GE Healthcare. Etoposide (E1383, Sigma) and bacterial LPS (L2880, Sigma) were purchased from Sigma-Aldrich. Glutathione-agarose beads and Halt Protease Inhibitor Cocktail was obtained from Thermo Scientific.