Ab199438

After washing twice with PBS, cells were lysed with RIPA buffer (1% NP-40, 0.1% SDS, 0.5% deoxycholate, 50 mM Tris [pH 7.4], and a protease inhibitor cocktail) on ice for 15 min, scraped with a scraper, and centrifuged at 12,000 rpm for 15 min at 4° C. After measuring the protein concentration with Bradford reagent (Sigma), the protein samples were adjusted, 6× loading buffer was added, and the samples were boiled for 5 min. Then, electrophoresis was performed. Then, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore). After incubation with TBST containing 5% skimmed milk (25 mM Tris, 150 mM NaCl, 0.05% Tween 20 [pH 7.5]) for 1 hour at room temperature, the primary antibody was incubated overnight with the membrane at 4° C. After washing the membrane three times with TBST, a horseradish peroxidase-conjugated secondary antibody was added, and the membrane was incubated for 1 hour at room temperature. The membrane was washed three times with TBST and developed with enhanced chemiluminescence (Pierce). Antibodies against GST (10000-0-AP), GAPDH (10494-1-AP), HA (51064-2-AP) and Flag (20543-1-AP) were obtained from Proteintech, and antibodies against LHX9 (ab224357), P53 (ab26) and PGK1 (ab199438) were obtained from Abcam.

Proteins were isolated using radioimmunoprecipitation assay (RIPA; Solarbio, Beijing, China) buffer with proteinase inhibitor. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then blotted onto the polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk, the membrane was incubated with the following primary antibodies overnight: anti-E-Cadherin (ab1416; Abcam, Cambridge, MA, USA), anti-Vimentin (ab92547; Abcam), anti-N-Cadherin (ab18203; Abcam), anti-HIF-1α (ab16066; Abcam), anti-glucose transporter 1 (anti-GLUT1; ab40084; Abcam), anti-phosphoglycerate kinase 1 (anti-PGK1; ab199438; Abcam), anti-pyruvate kinase M2 (anti-PKM2; ab137852; Abcam), and GAPDH (ab181602, Abcam) were used as the internal references for immunoblot. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) were used to incubate with the membrane the next day. Protein signals were visualized with the enhanced chemiluminescence detection kit (Millipore, Billarica, MA, USA).

Pretreated cells were gathered on ice and lysed with RIPA lysis buffer. Proteins were separated on 4%–12% Bis-Tris polyacrylamide gels and transferred to the PVDF membrane. The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. The following primary antibodies were adopted: Actin (Servicebio, GB12001; 1 : 1000); Aldoc (Proteintech, 14884-1-AP, 1 : 5000); CD74 (Bioss, bs-2518R, 1 : 2000); CYP51 (Proteintech, 13431-1-AP, 1 : 5000); EBP (Proteintech, 15518-1-AP, 1 : 1000); ENO2 (Servicebio, GB11376, 1 : 1000); FDFT1 (Proteintech, 13128-1-AP, 1 : 1000); FOXO1 (Servicebio, GB11286; 1: 2000); Gapdh (Proteintech, 60004-1-Ig, 1 : 20000); Gpi1 (Proteintech, 15171-1-AP, 1 : 1000); Hif-1a (Servicebio, GB111339, 1 : 1000); Mif (Proteintech, 26992-1-AP, 1 : 1000); NSDHL (Proteintech, 15111-1-AP, 1 : 1000); PFKL (abcam, ab181064, 1 : 5000); Pgk1 (abcam, ab199438, 1 : 2000); PKM (abcam, ab150377, 1 : 5000); p53 (Proteintech, 10442-1-AP, 1 : 1000); Tpi1 (abcam, ab196618, 1 : 1000).