Ecl chemiluminescence reagent

Cells were lysed in 1 × SDS protein loading buffer to obtain a total protein sample. The proteins separated on a 12% SDS polyacrylamide gel and blotted on a PVDF membrane. After blocking the 5% skim milk powder at room temperature for 2 h, the primary antibody was incubated overnight at 4°C, the secondary antibody was incubated at room temperature for 1 h, and then the PVDF membrane was washed 5 times with TBST. And visualized with ECL chemiluminescence reagent (Yeasen, Shanghai, China) using GeneGnome Chemiluminescence Imaging System (Gene, Hong Kong, China).

The plasmids encoding HA-MDM2, p53, His-Ub were described previously [34 ]. The Flag-tagged pENTER-UTP11 plasmid was purchased from Vigene Biosciences (Shandong, China). Human UTP11 and MPP10 were subcloned into the vectors of Flag-pcDNA3.1 and Myc-His-pcDNA3.1, respectively. The primers for subcloning are listed in Supplementary Table 7. The anti-Flag (Cat. No. F1804, Sigma-Aldrich, St louis, MO, USA), anti-HA (Cat. No. 2367, Cell Signaling Technology, Danvers, MA, USA), anti-UTP11 (Cat. No: #46701, Signalway Antibody), anti-p53 (Cat. No. sc-126, DO-1, Santa Cruz Biotechnology), anti-MDM2 (Cat. No. ab16895, 2A10, Abcam), anti-GAPDH (Cat. No. 60004-1-Ig, Proteintech), anti-RPL5 (Cat. No. ab86863, Abcam), anti-RPL11 (Cat. No. ab79352, Abcam), anti-p21 (Cat. No. 2947, Cell Signaling Technology), anti-B23 (Cat. No. sc-271737, Santa Cruz BioteChnology), anti-MPP10 (Cat. No. ER65428, Hangzhou HuaAn Biotechnology), anti-SLC7A11 antibody (Cat. No.26864-1-AP, Proteintech), anti-NRF2 (Cat. No.16396-1-AP, Proteintech), and anti-vinculin antibody (Cat. No.13901, Cell Signaling Technology) were commercially purchased. The secondary antibodies used were HRP-conjugated affinipure goat anti-rabbit IgG (Cat. No. SA00001-2, Proteintech) and anti-mouse IgG (Cat. No. SA00001-1, Proteintech). Proteins were visualized with the ECL chemiluminescence reagent (Yeasen).