Ab273721

The performance of western blot was conducted as some time ago published [25 (link)]. The antibodies were listed as follows: anti-AGK (ab137616; 1:500; Abcam, Cambridge, MA, USA), anti- Hexokinase 2 (HK2) (ab273721; 1:1,000; Abcam), anti-Lactate dehydrogenase A (LDHA) (ab101562; 1:500; Abcam).

Cells were collected in lysis buffer containing Complete Protease Inhibitor Cocktail (Roche) and applicated to SDS-PAGE. Cellular proteins were transferred onto a nitrocellulose membrane (Millipore) and probed for anti-OGDH (#ab307369, Abcam; dilution 1:1000), anti-LDHA (#sc-133,123, Santa Cruz Biotechnology; dilution 1:1000), anti-GPT (#sc-374,501, Santa Cruz Biotechnology; dilution 1:1000), anti-HK2 (#ab273721, Abcam; dilution 1:2000), anti-PDHA2 (#TA379757, OriGene Technologies, Inc.; dilution 1:1000), anti-PCK1 (#ab133603, Abcam; dilution 1:2000) or anti-GAPDH (#9484, Abcam; dilution 1:5000) antibodies. After washing for 3 times with PBS, the membranes were incubated with secondary antibodies. The reaction was measured using an ECL chemiluminescence kit (Amersham Biosciences) with Eastman Kodak Co. hyper film. The gel blots were quantified by Multi Gauge software (Fujifilm Life Sciences) and presented as the mean ± SD, based on at least three independent experiments.

Cells were lysed with RIPA buffer (Beyotime, China) for 15 min on ice and centrifuged at 10,000 x g for 15 min. The supernatant was collected, and the concentration of the protein sample was measured with the BCA kit (Beyotime, China). Protein samples were separated by electrophoresis and transferred from gel to PVDF membrane (Beyotime, China). 5% BSA in PBS was used to block the PVDF membrane, which was followed by the incubation with primary antibodies: anti-β-catenin (1 : 1000, ab32572, abcam), anti-CyclinD 1 (1 : 1500, ab141491, abcam), anti-C-myc (1 : 1000, ab32072, abcam), anti-HK2 (1 : 1500, ab273721, abcam), anti-LDHA (1 : 1000, ab290306, abcam), anti-E-cadherin (1 : 1000, ab233611, abcam), anti-N-cadherin (1 : 1000, ab254512, abcam), as well as anti-GAPDH (1 : 1500, ab8245, abcam) antibodies. The membrane was washed 3 times with TBST for 5 minutes each. After washing, the membrane was further incubated with HRP-linked secondary antibody (1 : 3000; #7047, Cell Signaling Technologies, MA, USA) at room temperature for 1 h. The protein bands were developed using the ECL protein detection kit (Solarbio, China) and analyzed with ImageJ software (NIH, USA).