Mycn sc 53993

Cell proteins were prepared by lysing cells for 30 min at 4⁰ C in a lysis buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin and 25 μg/ml leupeptin. Equal amounts of protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose filter. After blocking with buffer containing 5% non-fat milk, 20 mM Tris-HCl (pH 7.5), and 500 mM NaCl for 1 h at room temperature, the filter was incubated with specific antibodies for 1 h at room temperature; washed and then incubated with HRP-labeled secondary antibody; and developed using a chemiluminescent detection system (ECL, Amersham Life Science, Buckinghamshire, England). The specific antibodies used included MYCN (sc-53993, Santa Cruz), MDM2 (SMP14, Sigma; 2A10, Invitrogen), p53 (DO-1, Santa Cruz), HuD (AB5971, Sigma), ARID3B (A06737, Boster) and HMGA2 (PA5-25276, Invitrogen). All antibodies were used according to the manufacturers’ instruction.

ChIP assay was performed as previously described (10 (link), 30 (link)). The PCR primers used were: PKMYT1 promoter, 5′-TTATGGACCCAAACACTACGC-3′ and 5′-CGCCAAAAATTCCAAACC-3′; and BLM promoter, 5′-GGCTGAAACAGAAGCATGG-3′ and 5′-TCACCCGTACCCCTCTACAC-3′. Antibodies used in this study were: IgG (sc-2027, Santa Cruz Biotechnology), GAL4 (IgG2A negative control) (Santa Cruz Biotechnology), and MYCN (sc-53993, Santa Cruz Biotechnology).

Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate) in the presence of 1× protease and phosphatase inhibitor cocktails (Sigma-Aldrich, MO, USA). Proteins were resolved by SDS-PAGE and transferred to Hybond ECL membrane (GE Healthcare, CO, USA). The membrane was immunoblotted with antibodies against Poly(ADP-ribose) 10H (ALX-804-220-R100, 1:400, Enzo Life Sciences, NY, USA), PARP1 (sc-8007, 1:1000, Santa Cruz Biotechnology, TX, USA), MYCN (sc-53993,1:250, Santa Cruz Biotechnology) and TUBB (T8328, β-tubulin; 1:5000, Sigma-Aldrich), each diluted in 5% milk and incubated at 4°C overnight. After the application of the appropriate HRP-conjugated secondary antibody and further washes, the immunoreactive protein was visualised using ECL reagents (GE Healthcare, IL, USA) according to the manufacturer’s instructions.