Ab181981

Total protein in tissues and cells was extracted using RIPA buffer (Beyotime, Shanghai, China) and determined using a BCA Protein Quantification Kit (Vazyme). Twenty micrograms of proteins were separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Solarbio). Then the protein samples were transferred onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Thereafter, the membranes were blocked with non-fat milk for 1 h and probed with primary antibody: hexokinase2 (HK2; ab209847; Abcam, Cambridge, MA, USA), pyruvate kinase isozyme type M2 (PKM2; ab137852; Abcam), FLOT2 (ab181981; Abcam), or GAPDH (ab9485; Abcam) overnight at 4 °C. After incubation with secondary antibody (ab205719; Abcam) for 2 h at room temperature, the protein bands were examined by an enhanced chemiluminescence reagent (Vazyme).

We added 50 μg proteins from cell lysates to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred them to a polyvinylidene difluoride (PVDF) membrane (MilliporeSigma, Burlington, Massachusetts, US) for blotting with antibodies against CHK1(ab40866), IP10 (ab214668), Fas (ab133619) and Eg5 (ab181981; all from Abcam, Cambridge, UK); as well as with Phospho-CHK1-Ser317 (12302S), Phospho-CHK1-Ser345 (2348S) or BIM (2933T; all from Cell Signaling Technologies [CST], Danvers, Massachusetts, US). Additionally, we used anti-GAPDH (10494-2-AP), anti-BubR1 (11504-2-AP), anti-cyclin B1 (55004-1-AP), anti-UBE2S (14115-1-AP) and anti-p21 (10355-1-AP) from Proteintech (Rosemont, Illinois, US), as well as anti-MSX2 (A2017) from ABclonal (Woburn, Massachusetts, US) and anti-CENPF (DF2310) from Affinity Biotech (Cincinnati, Ohio, US), for immunoreactivity (overnight at 4° C) which was visualized with an enhanced chemiluminescence kit (MilliporeSigma).