Pcmv p16 ink4a

Sorafenib (Cat No: S-8502) was purchased from LC Laboratories. LY294002 (Cat No: L9908) and MTT (Cat No: M2003) were obtained from Sigma-Aldrich. ID1 overexpression plasmid pcDNA3 hId1(Cat No: #16061) and p16 overexpression plasmid pCMV p16 INK4A (Cat No: #10916) were purchased from Addgene. Antibodies against p-AKT (ser473) (Cat No: #4060) and p16 (Cat No: #2407) were purchased from Cell Signaling Technology. Antibodies against ID1 (Cat No: sc-488) and GAPDH (Cat No: sc-47724) were purchased from Santa Cruz Biotechnology. Neutralizing antibody of IL6 (Cat No: MAB206) was purchased from R&D Systems.

Myc-tagged Cullin 1, Cullin 2, Cullin 3, Cullin 4 A, Cullin 4B, Cullin 5, Cullin 7, βTrCP1, FBL17, FBXW7, CK1α1, CK1α2, CK1ε, CK1δ, CKIIα&β, HA-tagged CDH1, CDC20, RB1-WT, RB1-13A, RB1-13E, CBP, p300, E2F1, GSK3β S9A, S6K1 R3A, ERK1, MYC, CycA, and CycE. Flag-tagged IKKα EE, ΙΚΚβ S9E, and RB1, shRNA against Cullin 1, Cullin 2, CDK4, RB1, cyclin D1, cyclin D3, βTrCP1, CK1α, CK1δ, and CK1ε have been described previously in the papers published by the Wei laboratory^{28 (link),36 (link)}. Flag-tagged E2F4, E2F6, IRF3, DP1, MAX, FOXM1, and SP1 were purchased from OriGene. pCMV-p16 INK4A was purchased from Addgene. sgControl was a gift from Dr. W. Kaelin. sgRNAs targeting CDK4, CDK6, hCDK6 promoter driven luciferase reporter constructs, and RB1 mutation constructs were generated in this study. Primer sequence information used in this study has been provided in Supplementary Table 1.

pCMV-p16^INK4a was purchased from Addgene (Watertown, MA, USA.). HDF cells in six-well plates (5 × 10⁴/well) were transiently transfected with 2 μg of a vector or pCMV-p16^INK4a using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and incubated for 48 h. After that the cells were analyzed by immunoblotting method.