Anti jmjd2b

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Li et al., 2018d (link); Li Z. et al., 2019 (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Yang et al., 2019 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-p300 (Santa Cruz, sc-585), anti-c-Jun (Santa Cruz, sc-1694), anti-Fos (Santa Cruz, sc-166940), anti-SMAD3 (Abcam, ab28379), anti-ASH2 (Bethyl Laboratories, A300-489A), anti-JMJD2B (Bethyl Laboratories, A301-478), anti-anti-acetyl H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-449), anti-trimethyl H3K9 (Millipore, 07-441), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.

Cell extracts were collected and lysed using Radioimmunoprecipitation assay (RIPA) lysis buffer together with a protease inhibitor cocktail (Kangcheng, Shanghai, China). Proteins were separated using SDS-PAGE and then immunoblotted. The primary antibodies used in the present study were: anti-JMJD2B (catalog no. #A301-477A, 1:2000, Bethyl Laboratories, Montgomery, TX, USA); anti-phenylalanine hydroxylase (PAH) (catalog no. #sc-271258, 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-asparagine synthetase (glutamine-hydrolyzing) (ASNS) (catalog no. #14681-1-AP, 1:2000, Proteintech, Chicago, IL, USA); anti-histidine ammonia-lyase (HAL) (catalog no. # H00003034-M04, 1:2000, Abnova, Taipei, Taiwan); anti-α-tubulin (catalog no. #ab18251, 1:2000, Abcam, Cambridge, UK); anti-LC3B (catalog no. #2775, 1:1000), anti-cleaved Caspase 3 (catalog no. #9664, 1:1000), anti-cleaved Caspase 8 (catalog no. #9496, 1:1000), anti-cleaved Caspase 9 (catalog no. #9505, 1:1000), and anti-cleaved poly (ADP-ribose) polymerase (PARP) (catalog no. #5625, 1:1000) (all from Cell Signaling Technology (Danvers, MA, USA)). Secondary antibodies were conjugated with horseradish peroxidase (HRP) (Kangchen) and the signal was detected using an ECL Kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, Rockford, IL, USA).