Hrp conjugated goat anti mouse igg

After being washed with cold phosphate-buffered saline (PBS), cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biotechnology). The collected lysates were centrifuged at 12,000 ×g at 4 °C for 10 minutes, and then the total protein was quantified using a commercial bicinchoninic acid (BCA) kit (Biotechnology). Then, 30 µg of protein was loaded into each lane, and the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 8–12%), and transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore). Then, the membrane was blocked with skimmed milk and incubated overnight with the following antibodies: anti-TPD52 (1:10,000; Abcam), anti-Ki-67 (1:5,000; Abcam), anti-MCM2 (1:1,000; Abcam), anti-proliferating cell nuclear antigen (1:1,000; Cell Signaling Technology), and anti-GAPDH (1:10,000; Proteintech Group). After that, the membrane was washed 3 times with 1% tris-buffered saline (TBS) and the following secondary antibodies were added: horse-radish peroxidase (HRP) conjugated Goat Anti-Mouse IgG (1:5,000; Wuhan Boster Biological Technology) and HRP-conjugated Goat Anti-Rabbit IgG (1:5,000; Wuhan Boster Biological Technology). The protein bands were detected with a BeyoECL Plus kit (Biotechnology) and quantified by ImageJ.