H3k79me2

Cells were collected at 48 h post-transfection and proteins were extracted by cell lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). Protein samples were separated on 8–12% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, USA) using standard procedures. The membranes were blocked at room temperature for 2 h with 5% nonfat dry milk. Membranes were incubated with primary antibody against FLAG (1:2000, EneGene Biotech Co. Ltd, China), HIF-1α (1:1000, Novus Biologicals, Kittketon, Co, USA), H3K4me3 (1:5000, Abcam, UK), H3K79me2 (1:3000, Active Motif, Inc. Japan), GAPDH (1:5000, EneGene Biotech Co. Ltd, China), β-actin (1:100000, EnoGene Biotech Co. Ltd, China), and tubulin (1:10000, EnoGene Biotech Co. Ltd, China) overnight at 4°C. Membranes were washed 3 times for 10 min each and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:10000, KPL, Gaithersburg, MD, USA). The proteins were detected using enhanced chemiluminescence (ECL) reagents (Millipore, Billerica, MA, USA).

Immunoblots were developed using ECL Prime (Amersham RPN2232). Antibodies: H2B K123Ubq1 (Cell Signaling Technologies 5546; 1:1,000), H2B (Active Motif 39237; 1:5,000), H3K79me3 (Abcam 2621; 1:2,000), H3K79me2 (Active Motif 39143; 1:2,500), H3K4me3 (Epicypher 13-0004; 1:5,000), H3K36me3 (Abcam 9050; 1:1,000), H3 (Epicypher 13-0001; 1:5,000), G6PDH (Sigma Aldrich A9521; 1:100,000), FLAG (Sigma Aldrich F7425; 1:1,000). Rabbit (Amersham NA934; Donkey anti-Rabbit) and mouse (Amersham NA931; Sheep anti-mouse) secondary antibodies were used at 1:10,000.