Gsk 3β 27c10

After release from quiescence in the presence of DMSO or GUTK, cells were harvested and treated with ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail. Protein quantification, electrophoresis and western blotting were performed as previously described.^{42 (link)} Antibodies (human specific) used were c-MYC (Abcam, Epitomics, Cambridge, UK, #1472-1), S62-phospho-c-MYC (ab51156), T58-phospho-c-MYC (ab28842) and GAPDH (ab128915) were obtained from Abcam Company (Cambridge, UK). Additional antibodies were phospho-Rb (Ser807/811) (#9308), ERK1/2 (137F5) (#4695), phospho-ERK1/2 (Thr202/Tyr204) (#4370), GSK3β (27C10) (#9315) and phospho-GSK3β (Ser 9) (#9323) purchased from Cell Signaling Technology (Danvers, MA, USA), FBXW7 (A301-721A) were obtained from Bethyl Laboratories (Montgomery, TX, USA) and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Dallas, CA, USA).
Immunoblot images were exported in the format of tagged image file and quantified using ImageJ 1.46 software (National Institute of Health). After normalization to loading control, the immunoblot bands of c-MYC and FBXW7 were plotted to determine the half-life. The 50% decrease in protein intensity based on the Y-axis was used to determine the corresponding value on the X-axis, which by definition is the time required for the proteins of interest to decrease to the half of the baseline levels.

p-NF-κB [93H1], NFκB [D14E12], p-Akt Ser473 [D9E], Akt (pan) [C67E7], Cyclin D1 [92G2], p-Rb [D59B7], Rb [D20], GSK-3β [27C10], Caspase-3 [8G10], cleaved Caspase-3 [5A1E] from Cell Signaling, Danvers, MA; IL-33 (M-266) from Santa Cruz Biotechnology, Dallas, TX; GAPDH [GAPDH-71.1], Actin [A2228], Vinculin [hVIN-1] from Sigma Aldrich, St. Louis, MO; p-GSK-3β [75745] and nuclear matrix protein p84 [EPR5662(2)] from Abcam, Cambridge, MA. Immunohistochemical antibodies used were as follows: CD163 [EPR19518] from Abcam, Cambridge, MA and CD3 [1F4] from BD Biosciences, San Jose, CA.

PPI was purchased from the Institute for Drug Control (Shanghai, China, #111590) and dissolved in dimethyl sulfoxide (DMSO). Doxorubicin was purchased from Sangon Biotech (Shanghai, China, #DB3456) and dissolved in ultra-purified water. FITC Annexin V and propidium iodide were obtained from BD Biosciences (Franklin Lakes, USA, #556420). The cell cycle detection kit was from Key GENBioTECH (Nanjing, China). Antibodies against PARP (#9542), BAX (D2E11, #5023), BCL-2 (#2876), Vimentin (5G3F10, #3390), C-Myc (D84C12, #5605), GSK-3β (27C10, #9315), phosphor-GSK-3β (#8213), active β-catenin (D13A1, #8814) and β-catenin siRNA II (#6238) were from Cell Signaling Technology (Danvers, USA). Trypsin-EDTA, Lipofectamine 2000 (#11668) and TRIzol reagent (#15596) were from Invitrogen (Rockville, MD, USA). CHIR99021 (#SML1046) and antibody against β-actin (#A2228) were from Sigma-Aldrich (Saint Louis, USA). Monoclonal antibody of rabbit anti-Ki-67 and RIPA Cell Lysis Buffer (#P0013B) were got from Beyotime Institute of Biotechnology (Shanghai, China). SuperSignal Chemiluminescent HRP Substrate (#34078) was from Thermo Fisher scientific Inc (Rockford, USA). Matrigel (#356234) was purchased from BD Biosciences (Bedford, USA). PrimeScript RT reagent kit (#RR037A) and SYBR Premix Ex Taq II (#RR420L) were bought from TaKaRa (Dalian, China).