Appropriate secondary antibody

The protein precipitates were extracted using an RIPA protein lysate (cat no. 89900, Thermo Fisher) containing a protease inhibitor phenylmethylsulfonyl fluoride (cat no. 36978, Thermo Fisher, USA). Protein concentrations were determined using a BCA kit (ThermoFisher, USA). Then, the protein was subjected to SDS polyacrylamide electrophoresis and transferred onto a PVDF membrane (cat no. IPVH00010, Millipore). The PVDF membrane was blocked with 5% skimmed milk at room temperature. Then, the PVDF membranes were incubated overnight at 4 ^∘C with primary antibodies diluted with 5% skimmed milk. The used primary antibodies were mouse anti-PCNA monoclonal antibody (1:2000, cat no. ab29, Abcam, USA) and rabbit anti-p21 polyclonal antibody (1:1000, cat no. 10355-1-AP, Proteintech, Chian). Rabbit anti-vinculin monoclonal antibody (1:5000, cat no. ab129002, Abcam, USA) was used as the loading control. Then, the appropriate secondary antibody (CoWin Biosciences) was incubated with the PVDF membranes based on the genetic origin of the primary antibody. A gel imaging system was used to scan the protein bands using ECL reagents, and vinculin was used as an internal reference.

The cellular protein precipitates were extracted using a RIPA protein lysate containing a protease inhibitor Cocktail. Protein concentrations were determined using a BCA kit (ThermoFisher Scientific, USA). Then, the protein was subjected to SDS polyacrylamide electrophoresis and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% BSA at room temperature. Then, the PVDF membranes were incubated overnight at 4 °C with 5% skimmed milk and primary antibodies: AKT3 (Cell Signaling Technology), p-AKT (Cell Signaling Technology), and GAPDH (CoWin Biosciences). Then, the appropriate secondary antibody (CoWin Biosciences) was incubated with the PVDF membranes based on the genetic origin of the primary antibody. A gel imaging system was used to scan the protein bands using ECL reagents, and GAPDH was used as an internal reference.