Mouse anti mdm2

Proteins were lysed and extracted through incubation with radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich). Bradford reagent was used to quantify the extracted proteins. About 40 μg of protein were separated by Nupage 4%–12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Aurora, OH, USA). The membranes were blocked with 5% milk in phosphate-buffered saline with 0.05% Tween 20 (PBS-T) buffer and then incubated with the following primary antibodies: mouse anti-MDM2 (1:1000, Abcam, Cambridge, UK), p85 (1:1000 Cell Signaling), p53 (1:2500, Abcam, Cambridge, UK), p21 (Santa Cruz Biotechnology, TX, USA 1:1000), and rabbit anti-α-actin (1:2500, Sigma Aldrich), at 4 °C overnight. The membrane was washed with PBMS-T and then incubated for 1 h, at room temperature, with goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, GE Healthcare). The membranes were developed using a chemiluminescence reaction (ECL, GE Healthcare). The data were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

The indicated OV6⁺ HCC cells were seeded onto coverslips in 12-well plates and stained with rabbit anti-Flag (Cell Signaling Technology), anti-MDM2 (Santa Cruz Biotechnology) or mouse anti-MDM2 (Abcam) primary antibodies, followed by fluorescent staining with Alexa Fluor 555-conjugated IgG and Alexa Fluor 488-conjugated IgG (Invitrogen). Nuclear staining was conducted by 4,6-diamidino-2-phenylindole (DAPI). Representative images were captured with a Leica confocal microscope (Leica, Mannheim, Germany).

Proteins were extracted by incubation with RIPA buffer and quantified by Bradford reagent. Twenty-five micrograms of protein were separated on Nupage 4–12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride membranes (PVDF, GE Healthcare Life Science, Piscataway, NJ, USA) to be probed with the following antibodies: mouse anti-MDM2 (1 : 500, Abcam, Cambridge, UK); mouse anti-p21 (1 : 1000, Cell Signaling, Danvers, MA, USA), and rabbit anti-β-actin (1 : 5000, Sigma-Aldrich). For detection, goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (1 : 2000, GE Healthcare Life Science) were used. Signal detection was performed via chemiluminescence reaction (ECL, GE Healthcare Life Science). WB quantification was performed using ImageJ software analysis (National Institutes of Health, Bethesda, MD, USA).