Phosphor cdk2 t160

The following antibodies were used in the study: phospho-Rb T821 (Thermo Fisher Scientific, Waltham, MA, USA, 1:1000 dilution), Rb (Abcam, Cambridge, UK, 1:2000 dilution), phosphor-CDK2 T160 (Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), CDK2 (Abcam, Cambridge, UK, 1:1000 dilution), Cyclin E1 (Proteintech, Wuhan, China, 1:1000 dilution), p53 (Proteintech, Wuhan, China, 1:5000 dilution), MDM2 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:1000 dilution), and β-actin (Beyotime, Shanghai, China, 1:1000 dilution).
PF-07104091 and Compound III-13 were synthesized by WuXi AppTec (Shanghai, China).

Whole-cell lysates were prepared in RIPA Lysis buffer (Boston Bioproducts), containing protease and phosphatase inhibitors. Protein concentration was determined by BCA protein assay (Pierce) and equal amounts (25 μg) were subjected to SDS-PAGE. Western blot analysis was performed using monoclonal antibodies against CDK1, CDK4, CDK6, cyclin E (Santa Cruz Biotechnology) Cyclin D3, total S6 ribosomal protein, total RB, p27^Kip1 (Cell Signaling Technology) and actin (Genscript) and polyclonal antibodies against CDK2, CDK4, p21^Waf1/Cip1, pAKT, RSK1/2 (Santa Cruz Biotechnology), Cyclin D1, phospho-RB S807/811, phospho-RB T821, phospho-ERK1/2, total ERK1/2, total AKT, phospho-S6 ribosomal protein, phospho-4E-BP1, total 4E-BP1, total TSC2, phospho-TSC T1462, C-Raf, phospho-C-Raf, MEK1/2, phospho-MEK1/2, phospho-RSK1, E-cadherin, vimentin, N-cadherin, CDK7, phosphor-CDK2 T160 (Cell Signaling Technology) and phospho-TSC S1798 (Abgent).