Protein Extraction from Cancer Cells

The protein extraction protocol from cancer cells was previously reported [30 (link)]. Briefly, cells were collected and lysed using Radio Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime) with a protease inhibitor cocktail (Roche, IN, USA). Equal amounts of protein lysate were quantified using BCA kit (Thermo Fisher, USA), separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then sealed with 5% non-fat milk for 1 hour, and incubated with primary antibodies (Abcam, Cambridge, UK) against: FUS (ab84078, 1/1000), SLC10A1 (ab131084, 1/1000), β-actin (ab8227, 1/1000), GLUT1 (ab14683, 1/2500), HK2 (ab227198, 1/5000), LDHA (ab47010, 1/1000), PGK1 (ab154613, 1/1000) overnight at 4°C, with GAPDH (ab9485, 1/2500) as control. After washing, the membrane was incubated with goat anti-rabbit secondary antibody (ab96899, 1/1000, Abcam, Cambridge, UK) at 37°C for 1 hour. The protein bands were finally detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.