Supplemental Data The Signaling Adaptor p62 Is an Important NF-κB Mediator in Tumorigenesis

Supplemental Experimental Procedures Reagents and antibodies Reagents were purchased as follows: DRB, wortmannin, NAC, doxycycline, BHA, polybrene and H2O2 were from Sigma, and PD98159 from Calbiochem. Polyclonal anti-phospho-AKT, anti- AKT, anti-phospho-S6K, anti-phospho-ATF2, anti-ATF2, anti-phospho-p38, anti-p38, anti- phospho-IKKα/β and anti-JNK antibodies were from Cell Signalling. Anti-actin, anti-phospho- JNK, anti-IKKβ, anti-IKKγ, anti-TRAF6, anti-UBI, anti-phospho-c-Jun, and anti-c-Jun antibodies were purchased from Santa Cruz. Polyclonal anti-p62 C-ter was from Progen and human monoclonal anti-p62 was from BD. Anti-Ras was from Calbiochem. All antibodies were used according to manufacturers' instructions. Cell culture WT and p62 KO primary EFs were derived from E13.5 embryos (Garcia-Cao et al., 2003). Cells were maintained in DMEM (Gibco BRL) supplemented with 10% (v/v) fetal calf serum (FCS), 1% glutamine and 1% penicillin/streptomycin (Gibco-Invitrogen) in an atmosphere of 95% air and 5% CO2, and immortalized by retroviral infection with pBabeT-Ag followed by puromycin selection (1µg/ml). The established cell lines represent pools of at least 100 independent clones. The HEK293-derived virus packaging cell line 293T, HeLa cells and all human cancer lines were cultured in DMEM with 10% FCS. For knockdown of p62 in human cancer cell lines, two different human siRNAs for p62 were obtained from Qiagen with the following target sequences: TAGGGTGCAAGAAGCCATTTA and CTCATAGGTCCCTGACATTTA. AllStars Negative Control siRNA (Qiagen) was used as negative control. Transfection of sRNAi was performed by calcium phosphate method. Reporter κB assays were performed as described (Sanz et al., 2000) using Renilla to normalize transfection efficiency. Transfection of EFs for κB assays and for RelA immunofluorescence was performed by Fugene, following manufacturers' instructions. For immunofluorescence, The different p62 promoter deletions were subcloned into pGL3-Basic vector (Promega). A point mutation to disrupt the AP1 enhancer element in the p62 promoter was introduced by Site Directed Mutagenesis (Stratagene): AP1 site (TGACTCA) was mutated to AP1 mut (TCTGTCA).