Anti ldha 19987 1 ap

The following reagents were used in this study: lipofectamine 2000 (11668019; Invitrogen), polybrene (TR-1003; Sigma-Aldrich), actinomycin D (S8964; Selleck), DAPI (H1200; Vector Laboratories), complete EDTA-free protease inhibitor cocktail (K1007; APExBIO), protein A/G agarose beads (20421; Thermo Fisher Scientific), anti-Flag M2 affinity agarose beads (A2220; Sigma), and streptavidin agarose resin (20349; Thermo Fisher Scientific).
The following antibodies were used in this study: anti-HK2 (22029-1-AP), anti-GPI (15171-1-AP), anti-ALDOB (18065-1-AP), anti-PGK1 (17811-1-AP), anti-PGAM1 (16126-1-AP), anti-ENO1 (11204-1-AP), anti-LDHA (19987-1-AP), and PABPC1 (10970-1-AP) antibodies were purchased from Proteintech. Anti-GFP (sc-9996), anti-β-actin (sc-47778), and anti-GAPDH (sc-166545) antibodies were purchased from Santa Cruz Biotechnology. Anti-c-Myc (9402 S), anti-PKM2 (4053 S), and anti-ubiquitin (3936 S) antibodies were purchased from Cell Signaling Technology. Anti-PFKL (ab181064) antibody was purchased from Abcam. Anti-Flag (F3165) and anti-MKRN3 (HPA029494) antibodies were purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated secondary antibodies against rabbit (111-035-144) and mouse (115-035-062) were purchased from Jackson ImmunoResearch.

Cell and tissue were lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails (NCM Biotech, China) for 30 min at 4°C and then centrifuged at 13000 × g for 30 min. Protein supernatants were separated by 7.5% (wt/vol) SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, USA). After blocking with 5% milk for 1 h at RT, the membranes were incubated with the primary antibodies at 4°C overnight and then with secondary antibodies conjugated to a fluorescent tag (Invitrogen, USA). The band signals were visualized by an Odyssey Infrared Imaging System (LI-COR, USA). The following primary antibodies were used in the study: anti-ALKBH3 (ab251697, Abcam, USA), anti-LDHA (19987–1-AP, Proteintech, China), anti-LDHB (14824–1-AP, Proteintech, China), anti-SP100 (PA5-53476, ThermoFisher, USA), anti-PML (ab179466, Abcam, USA), anti-YTHDF1 (57530, CST, USA), anti-Pan Kla (1401RM, PTM, China), anti-H3K18la (1406RM, PTM, China), anti-Flag (2368S, CST, USA), anti-ACTB (3700, CST, USA) and anti-Histone H3 (4499, CST, USA). Unprocessed western blot figures were performed in Supplementary Figures S14 and S15.

Cells were lysed on ice for 30 min in RIPA lysis buffer supplemented with protease inhibitors and a phosphatase inhibitor cocktail (Bimake), followed by centrifugation at 12,000 g for 10 min. The protein concentration was measured using a BCA Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The immunoblots were visualized by ECL chemiluminescence (GE healthcare, Buckinghamshire, UK) using a Bio-Rad gel image analysis system. The following primary antibodies were used in this study: anti-ETV4 (sc-113, Santa Cruz), anti-HK2 (22029-1-AP, Proteintech), anti-LDHA (19987-1-AP, Proteintech), anti-PDK1 (3062 T, Cell Signaling Technology), anti-c-MYC (10828-1-AP, Proteintech), anti-OCT4 (11263-1-AP, Proteintech), anti-NANOG (14295-1-AP, Proteintech), anti-LIN28 (11724-1-AP, Ptoteintech), anti-SHH (ab53281, Abcam), anti-GLI1 (66905-1-Ig, Proteintech), anti-CXCR4 (60042-1-Ig, Proteintech), anti-β-actin (A1978, Sigma-Aldrich).