Mabe462

ChIP was performed using a ChIP assay kit (Millipore) following the manufacturer’s instructions. DNA fragments, produced by sonication, were incubated with antibodies against TET2 (MABE 462, Millipore, USA) and IgG (as a control) at 4 °C overnight. Enriched DNA fragments were detected by PCR with the following FBP1 ChIP primers: F:5′-GATCCCCGACCTTGTCTGAA-3′, R:5′-TCGCGGAAACCTTTAGACGC-3′.

Cells were lysed and boiled for 10 min in sample buffer (2% SDS, 10% glycerol, 10% β-Mercaptoethanol, Bromphenol Blue and Tris-HCl, pH 6.8). Equal amounts of protein (50–100 μg) from cell lysate were denatured in sample buffer (Thermo Fisher Scientific), subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific). Primary antibodies were against AR (dilution 1:1000; #sc-816, Santa Cruz Biotechnology), CXXC5 (dilution 1:1000; #16513-1-AP, Proteintech), CXXC4 (dilution 1:500; #ab105400, Abcam), TET2 (dilution 1:1000; #MABE462, Millipore), TET3 (dilution 1:1000; #ab139311, Abcam), TET1 (dilution 1:1000; #ab191698, Abcam), ID3 (dilution 1:500, #sc-56712, Santa Cruz Biotechnology), PFN2 (dilution 1:1000; #sc-100955, Santa Cruz Biotechnology), BRD4 (dilution 1:1000; #ab128874, Abcam), p300 (dilution 1:1000; #MS-586-PO, Thermo Scientific), ID1 (dilution 1:1000; #ab66495, Abcam), FOXA1 (dilution 1:1000; #ab23738, Abcam), Flag (dilution 1:1000; #F1804, Sigma Aldrich) and V5 (dilution 1:1000; #A190-120A, Bethyl Laboratories) and ERK2 (dilution 1:2000; #sc-1647, Santa Cruz Biotechnology).

For western blot, proteins were separated on a denaturing SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked in PBST +5% nonfat dry milk at room temp for >10 min or at 4°C overnight. Primary antibodies used for western blot were: FLAG M2 monoclonal antibody (Sigma Aldrich F1804), TET2 monoclonal antibody (Millipore MABE462), TET3 polyclonal antibody (Millipore ABE383), OGT polyclonal antibody (Santa Cruz sc32921), OGT monoclonal antibody (Cell Signaling D1D8Q), His6 monoclonal antibody (Thermo MA1-21315), JL8 GFP monoclonal antibody (Clontech), and O-GlcNAc RL2 monoclonal antibody (Abcam ab2739). Secondary antibodies used were goat anti-mouse HRP and goat anti-rabbit HRP from BioRad. Blots were incubated with Pico Chemiluminescent Substrate (ThermoFisher) and exposed to film in a dark room.