Anti ring1b

The lysates of BM cells (2 × 10⁶) from 8-week-old mice were prepared for the ChIP assay^{70 (link)}. BM cell lysates were sonicated and immunoprecipitated with the anti-Bmi1 (Santa Cruz Biotechnology, Inc. sc-10745, 2 μg sample⁻¹), anti-Cbx7 (Santa Cruz Biotechnology, Inc. sc-70232, 2 μg sample⁻¹), anti-H2AK119ub (Cell Signaling Technology 8240, 2 μg sample⁻¹), anti-H3K9ac (Merck 06-942, 2 μg sample⁻¹), anti-H3K4me3 (Abcam ab12209, 2 μg sample⁻¹), anti-H3K27me3 (Merck 07-449, 2 μg sample⁻¹), anti-Phc2 (Santa Cruz Biotechnology, Inc. sc-160664, 2 μg sample⁻¹), and anti-Ring1b (Cell Signaling Technology 5694, 2 μg sample⁻¹) Abs. Additionally, GSK126-treated OP9 cell lysates were sonicated and immunoprecipitated with the anti-Bmi1, anti-Phc2, and anti-Ring1b Abs. After immunoprecipitation, immune complexes were collected with protein A agarose (Merck GE17-0963-03) and extracted with an extraction buffer (1% SDS, 0.1 M NaHCO₂). DNA cross-links were reversed by heating to 65 °C for 8 h. DNA was extracted with phenol/chloroform and precipitated with ethanol. DNA isolated from an aliquot of total nuclear extract was used as the loading control for PCR (input control). qPCR was performed as described above. Data are presented after normalizing each immunoprecipitated DNA Ct value to 10% of the input DNA Ct value.

Cells were lysed in RIPA buffer. Cell lysate samples were resolved by SDS-PAGE and blotted to PVDF membranes. The blots were probed with anti- FLAG (Sigma), anti-BCOR (Bethyl Laboratoris), anti-BCL6 (Cell Signaling), anti-PCGF1 (Abcam), anti-RING1B (Cell Signaling), and anti-phospho-AKT (Cell Signaling), followed by anti-rabbit HRP-conjugated antibody (Bio-Rad). Immunostained proteins were detected by ECL (Amersham Pharmacia Biotech). Additional details are provided in supplementary methods (Additional file 1).

Wild type and RING1B-HaloTag cells were passaged together in a 1:1 ratio. Cells were trypsinised and labelled with Halo-TMR in suspension as described for fluorescence-based protein quantification above. Labelled cells were fixed with 3.3% formaldehyde for 10 min at room temperature. Permeabilisation (0.5% Triton X-100 in PBS), blocking (3% BSA in PBS for 30 min), primary (2.5 h, anti-RING1B, Cell Signalling Technology) and secondary antibody incubations (1 h, Supplementary Table 3) and DAPI incubation (0.1 μg/ml, 10 min) were all performed in suspension. Following all treatments, cells were resuspended in VECTASHIELD mounting medium (Vector Laboratories), and 10 μl of cells were flattened on a slide by applying pressure to a coverslip. Single Z-slices of nuclei were acquired using the above described spinning disk confocal system with an Olympus PlanApo 60x/1.4 N.A. oil-immersion objective and Volocity software (PerkinElmer). Signal from Halo-TMR in the 561 nm channel was used to differentiate wild type and RING1B-HaloTag cells. Signal from secondary antibody was acquired using the 488 nm laser at 10% laser power with 500 ms exposures. Nuclei were segmented manually and mean 488 nm channel fluorescence calculated for each cell line.

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).