Western Blot Analysis of AFF4 and GAPDH

Proteins were extracted using a radioimmunoprecipitation assay (Boster, China), and protein content was estimated using the bicinchoninic acid assay (Boster). They were electrophoresed in 10% sulfate-polyacrylamide gel. Subsequently, the proteins were transferred onto polyvinylidene fluoride membranes, washed with Tris-buffered saline with Tween 20, and blocked with 5% BSA blocking buffer at 37°C for 2 h. Then, primary antibodies, AFF4 (ab103586, Abcam, UK) and GAPDH (ab8245, Abcam), were incubated with the membranes overnight at 4°C, prior to incubation with secondary antibody (ab6721, Abcam) for 1 h at 37°C. The ECL detection kit (Yeasen, China) and ImageJ software (Version 1.48; NIH, USA) were used to visualize and determine the protein levels [27 (link)].