β action

Total protein extracted from cells was placed in radioimmunoprecipitation assay buffer (Vazyme Biotech Co., Ltd., Nanjing, China) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland), and protein concentrations were measured with the protein BCA assay kit (Invitrogen; Thermo Fisher Scientific). An equal amount of total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Vazyme Biotech Co., Ltd.) and transferred to polyvinylidene fluoride membrane (Invitrogen; Thermo Fisher Scientific). After blocking with 5% nonfat dried milk, the membrane was incubated with primary antibody against AFF4 (1:1000, Cell Signaling Technology, MA, USA) and β-action (1:5000, Cell Signaling Technology), followed by horseradish peroxidase-conjugated secondary antibody. Protein detection was visualized with the enhanced chemiluminescent detection reagent (Pierce; Thermo Fisher Scientific) in LI-COR imaging system (LI-COR Biosciences, NE, USA). Protein expression levels were quantified by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA) and normalized to β-action.

Thirty mg protein extracted from there OS cell lines and samples were used for immunoblotting as described elsewhere [21 (link), 22 (link)]. All the antibodies employed in our experiment were displayed in Supplementary Table 1. β-action (1:1000, cell signaling technology) was used as an internal reference. This test was repeated in triplicate. Immunofluorescence was performed as described elsewhere. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). All the antibodies employed in our experiment were displayed in Supplementary Table 1.