C myc

Antibodies used in Western blots were: Actin (sc-1616), GAPDH (sc-25778), IκBα (sc-371), p65 (sc-8008), Bcl-3 (sc-185) and P-c-Myc (sc-8000) (Santa Cruz Biotechnology); HA (3724), Ubiquitin (3936), P-Akt (4068), P-IκBα (2859), P-JNK (4668) and P-p65 (3033) (Cell Signaling Technology); IL-6 (9324) (R&D Systems); CYLD (SAB4200061, Sigma-Aldrich); c-Myc (626802, Biolegend); TNF-α (654250, Calbiochem); IKKγ (IMG-5480-2, Novus Biologicals); K63-Ubiquitin (ab179434), p19 (ab80) and p16 (ab51243) (Abcam) and γH2AX (05-636) (Millipore). For immunoprecipitation 300 µg cell lysate were incubated at 4ºC overnight. Then washed, and performed the immunoblotting.

Protein extracts were obtained from pieces of tumors or from HaCaT cells. Total protein extracts (30 μg) were subjected to SDS/PAGE. The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany). In all cases samples were subjected to luminography with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Illinois, USA).

Total protein extracts (40 μg) were subjected to SDS/PAGE. The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL, USA; BioRad, Marnes-la-Coquette, France) and probed with antibodies against IKKα (NB100-56704) (Novus Biologicals, Cambridge, UK); c-Myc (626802; Biolegend, San Diego, CA, USA); Actin (6276; Abcam, Cambridge, UK); GAPDH (sc-32233; Santa Cruz Biotechnology Inc. Europe, Heidelberg, Germany); MMP9 (AB19016; Merck Millipore, Darmstadt, Germany); Bcl2 (3498), cleaved-Caspase 3 (9661), Akt (4691) and P-p65 (3039) (Cell Signaling Technology, Danvers, MA, USA); Cyclin D1 (MA5-16356; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). In all cases, samples were subjected to luminography with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc., Rockford, IL, USA) or with Clarity Western ECL Substrate, 1705061 (BioRad, Marnes-la-Coquette, France).