Anti mycn b8.4 b sc 53 993

Cells were lysed for Western blotting in RIPA buffer (Cell Signaling Technology) with complete protease inhibitor cocktail (Roche). For the detection of histone H3 acetylation levels, cells were lysed in SDS lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% (v/v) glycerol, 1 mM DTT and complete protease inhibitor cocktail). The following antibodies were used: anti-MYCN (B8.4.B, sc-53,993, Santa Cruz), anti-ELOVL2 (EPR11880, ab176327, Abcam), anti-monoubiquitin H2A (Lys119) (ABE569, Millipore), anti-BMI1 (6964, Cell Signaling Technology), anti-RING1A (2820, Cell Signaling Technology), anti-RING1B (5694, Cell Signaling Technology), anti-SREBP1 (sc-13,551, Santa Cruz Biotechnology), and anti-ß-actin (clone AC-15, Sigma-Aldrich).

A total of 1 × 10⁶ cells were collected and lysed for ChIP in buffer containing 50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA and complete protease inhibitor cocktail (Roche), then sonicated to obtain 200–1000 bp DNA fragments. ChIP was performed according to the ChIP Assay Kit (Millipore) protocol. The following antibodies were used: anti-MYCN (B8.4.B, sc-53,993, Santa Cruz), anti-acetyl-histone H3 (Lys9) (7–352, Millipore), anti-trimethyl-histone H3 (Lys4) (07–473, Millipore), anti-monoubiquitin H2A (Lys119) (ABE569, Millipore), anti-BMI1 (6964, Cell Signaling Technology), anti-RING1A (2820, Cell Signaling Technology), anti-RING1B (5694, Cell Signaling Technology), anti-SREBP1 (sc-13,551, Santa Cruz Biotechnology), normal mouse IgG (sc-2025, Santa Cruz) and normal rabbit IgG (sc-2027, Santa Cruz). Primers for specific qRT-PCR amplification of the ELOVL2 promoter region were: forward: 5′-ATCAGTTCGGATAACGGCCC-3′, reverse: 5′- TAGAAGCGCAGGCTCTAGGA-3′.
ChIP sequencing data of ELOVL2 promoter interactions with MYCN and H3K4me3 in NGP, SK-N-BE and SK-N-SH NB cell lines were achieved from Gene Expression Omnibus (GSM2113529, GSM2113519 and GSM2308437), and the analysis was performed online in the Cistrome platform (http://cistrome.org).