Anti myc n 262

Crude nuclear protein extract from Nutlin-3a-treated (10 μM for 4 h) IMR-32 cells and Hela cells were prepared. Nuclei were isolated using hypotonic NP-40 buffer (10 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF. cOmplete Protease Inhibitor Cocktail, and 0.2% NP-40) and lysed via sonication in nuclei lysis buffer (50 mM Tris HCl pH 8.00, 150 mM NaCl, 1 mM DTT, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, cOmplete Protease Inhibitor Cocktail, and 0.05% NP-40). One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C. Recombinant Protein G agarose resin (Invitrogen) was used to purify protein complexes. Following three washes with 0.15% NP-40 in nuclei lysis buffer, protein samples were eluted by boiling resin in Laemmli sample buffer, separated using SDS-PAGE, and analyzed using Western blot with an anti-MYCN monoclonal antibody (B8.4.B, Santa Cruz Biotechnology), anti-p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology), anti-MAX antibody (C17, Santa Cruz Biotechnology), and anti-MYC (N262, Santa Cruz Biotechnology).

ChIP assays were carried out according to manufacturer's instructions (ChIP-IT Express Enzymatic Kit, Active Motif). The following antibodies were used: anti-MYC (N-262, Santa Cruz) or anti-MIZ1 (N-17, Santa Cruz). For the amplification of R1, R2 and R3 regions, the following previously reported primers were used: 5′-AGCAGGCTGTGGCTCTGATT-3′ (R1, Forward), 5′-CAAAATAGCCACCAGCCTCTTCT-3′ (R1, Reverse), 5′-ACCGGCTGGCCTGCTGGAACT-3′ (R2, Forward), 5′-TCTGCCGCCGCTCTCTCACCT-3′ (R2, Reverse), 5′-TCTGTCTCGGCAGCTGACAT-3′ (R3, Forward), 5′- ACCACAAAAGATCAAGGTGAGTGA-3′ (R3, Reverse). Each sample was used as template in Real-Time PCR to evaluate the enrichment of R1, R2 or R3 regions.