Sp60025

Antibodies against TRAF1, TRAF2, TRAF3, TRAF6, p50, p52, p105, p100, IκBα, the phosphorylated forms of IKKα (Ser176) β (Ser177), p65 (Ser536), c-jun (Ser63) and IL-6 were purchased from Cell Signaling Technology. Antibodies against p65 and TRAF5 were obtained from Santa Cruz Biotechnology. The antibody against EVER2 was purchased from Interchim. The anti-lamin B antibody was obtained from Thermo Scientific. Recombinant human TNFα was purchased from Miltenyi Biotec. Anti-actin, anti-Flag and anti-hemaglutinin (HA) antibodies, PMA, ionomycin, brefeldin A, Ly294002, SP60025, Bay 11-7082, Gö6976, AG1478, Akt1/2 kinase inhibitor and N-acetylcysteine were obtained from Sigma-Aldrich. Anti-Gluc and anti-Gfp antibodies were obtained from BioLabs. Anti-KL1 antibody was purchased from Immunotech Biotechnologies. Anti-ubiquitin antibody was obtained from Dako.

Subconfluent cells were cultured in respective complete growth medium in the presence of cetuximab (0.1, 1 or 10 μg ml⁻¹; Merck serono), rituximab (0.1, 1 or 10 μg ml⁻¹; Roche), rapamycin (50 nM; Calbiochem), PD98059 (50 μM; Sigma-Aldrich), GW5074 (20 μM; Sigma-Aldrich), LY94002 (25 μM; Sigma-Aldrich) or SP60025 (20 μM; Sigma-Aldrich). Medium was changed every 2–3 days. After at least 72 h, cells were stimulated as indicated and target gene expression was assayed by RT–qPCR or western blotting.