Enhanced radioimmunoprecipitation assay lysis buffer

Cells from each culture group were detached by trypsin, collected, and lysed with an enhanced radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology Co., Ltd.) containing a protease inhibitor. After measurement of protein concentration with a bicinchoninic acid (BCA) quantitation kit (Boster Biological Technology), samples of protein were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% bovine serum albumin (BSA) at room temperature for 1 hour and added with diluted primary antibodies (antibody to KDM5B, ab181089; Abcam; antibody to YTHDF3, Cat # 25537‐1‐AP; Proteintech; antibody to ITGA6, Cat # 3750; CST) for incubation overnight at 4°C. The following day, the membrane was washed 3 times with Tris‐buffered saline Tween‐20, re‐probed with horseradish peroxidase (HRP)‐labelled secondary antibody of goat anti‐rabbit for 1 hour at room temperature and developed with enhanced chemiluminescence working solution (EMD Millipore). Finally, ImageJ analysis software was used to quantify the grey levels of each band in the Western blot image normalized to GAPDH.

The total protein content was isolated with an enhanced radio immunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After being blocked in sealing solution, the membrane was incubated with the primary antibodies rabbit anti-human CCNE1 (1:2000, ab33911), CCNE2 (1:500, ab32103), KI67 (1: 000, ab92742), proliferating cell nuclear antigen (PCNA) (1:1000, ab925522), or GAPDH (1:5000, ab181602, all from Abcam Inc., Cambridge, MA, USA), which served as a NC, at 4 °C overnight. The next day, the membrane was incubated with secondary goat anti-rabbit IgG (1:10000, ab205718, Abcam Inc., Cambridge, MA, USA) at 37 °C for 1 h. The samples were developed using ECL reaction solution, photographed using SmartView Pro 2000 (UVCI-2100, Major Science, Saratoga, CA, USA), followed by gray scale analysis of the protein band pattern using the Quantity One software.