Ab3479

Chromatin immunoprecipitation was performed as previously described by Schödel et al. (14 (link)) using antibodies against HIF1A (PM14), EPAS1 (PM9) (9 (link),48 (link)), ARNT (Novus, NB-100–110; RRID:AB_10003150), SIN3A (Abcam, ab3479; RRID:AB_303839) and a Rabbit Control IgG (Abcam, ab46540; RRID:AB_2614925).
Libraries were prepared using the Illumina ChIP-seq kit and sequenced on the Illumina GAII platform according to the manufacturer’s protocol. Sequences were mapped to the GRCh37/hg19 assembly of the human genome using ELAND software (HIF ChIPseq) or Bowtie2 (REF) (SIN3A ChIPseq). Binding peaks were determined from the aligned reads using MACS software (49 (link)) using default parameters and the pre-immune sample as a control. A single ChIP experiment was analyzed by ChIP-sequencing and Raw and mapped data are available at GEO (GSE89836 and GSE103245). The analysis of mapped reads and genomic intervals defined by the bound regions was performed with the GenomicRanges (50 ) and Genomation (51 (link)) Bioconductor packages.

Western blots were performed as described before¹² (link) using antibodies listed in Table S2. Briefly, ESCs were cultured on feeder-free gelatin-coated plates for 1 day and protein was extracted using RIPA buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 2% Nonidet-P40, 2.5 mM EDTA, 0.1% SDS, 0.5% DOC) supplemented with Halt PIC (Thermo, 78430) and quantified with BSA assay (Thermo Scientific 23227). 20 μg of protein lysate was mixed with 2x Laemmli buffer and resolved on 7-9% SDS-PAGE gel (Mini-PROTEAN electrophoresis chamber, Bio-Rad), and transferred to PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad) in 10% methanol transfer buffer following the manufacturer’s instructions. Membranes were blocked in 5% milk in PBS with 0.1% Tween-20 (PBS-T) and incubated overnight at 4°C with primary antibodies (anti-Tet2 1:1000 Abcam ab124297; anti-Tet1 1:3000 GeneTex, GTX125888; anti-Vinculin 1:1000 Proteintech 66305; anti-Flag 1:1000 Sigma M2 F1804; anti-Sin3a 1:1000 Abcam ab3479; anti-beta actin 1:30000 Abcam ab6276). Next day, membranes were washed twice with PBS-T (10min each) and incubated with secondary antibody (goat anti-mouse HRP 401253, or goat anti-rabbit HRP, 401393, Millipore 1:2500) for 1 hr at room temperature. Protein bands were detected using ECL chemiluminescence reagent (Amersham RPN2106) and standard radiography (Konica SRX-101A).

Western blotting analysis were performed in accordance with standard procedures. The antibodies used in this study were NKX2–8 (1:500, Abcam, ab125040), CPT1A (1:1000, Abcam, ab128568), CPT2 (1:1000, Abcam, ab181114), Sin3A (1:1000, Abcam, ab3479), SAP18 (1:1000, Abcam, ab31748), HDAC1 (1:2000, Abcam, ab7028), p-AKT1 (phospho S473) (1:500, Abcam, ab81283), p-mTOR (phospho S2448) (1:1000, Abcam, ab109268), p-S6K1 (phospho T389) (1:1000, Abcam, ab60948), p-Bad (phospho S112) (1:2000, Abcam, ab129192), activated caspase-3 (1:500, Abcam, ab2302) and Cytochrome C (5 μg/ml, Abcam, ab13575). Anti-α-Tubulin antibody (1:3000, Abcam, ab7291) was used as a loading control.