Recombinant gsk 3β

Cycloheximide, actinomycin D, MG132, insulin, LiCl, SB415286 and DAPI were purchased from Sigma-Aldrich. The restriction endonucleases (NheI, XhoI, XbaI, EcoRI, KpnI and BamHI) and recombinant GSK-3β were bought from New England Biolabs. All of the primers used were synthesized from Integrated DNA Technologies. Protein A/G plus agarose was purchased from Santa Cruz. The anti-HA, anti-Myc antibodies and SignalSilence Control siRNA, SignalSilence GSK-3α/β siRNA were obtained from Cell Signaling; anti-actin was obtained from Sigma-Aldrich; anti-SCF^Fbw7 was obtained from Abcam and anti-SREBP-1 was bought from Becton-Dickinson and Co. GenJet Plus transfection reagent was purchased from SignaGen Laboratories and Halt™ combined protease and phosphatase inhibitor cocktails were bought from Thermo Scientific. Trypsin and Lys-C enzymes for mass spectrometry and the Dual-Luciferase® Reporter (DLR™) Assay System were obtained from Promega. SCF^Fbw7 siRNA 1 (s30664), SCF^Fbw7 siRNA 2 (s224357), SimplyBlue and Lipofectamine RNAiMAX were purchased from Invitrogen. AdEasy XL Adenoviral Vector System was purchased from Agilent Technologies.

Recombinant GSK3β (New England Biolabs) was diluted to 20 ng/μl in kinase buffer and incubated with 4 μg of GST-IRF1 or GST. The final concentration of ATP was 250 μM, in 25 μl. The reaction was carried out at 37°C for 1 h, and terminated by the addition of 6 μl of 5× Laemmli buffer (with 100 mM DTT). Samples were separated by SDS-PAGE and immunoblotted with indicated antibodies.

The RCC cell lines ACHN, Caki1, and A498 were obtained from American Type Culture Collection (Manassas, VA, USA). ACHN is derived from pleural effusion in metastatic RCC having wild type of VHL [23 (link), 24 (link)]. Caki1 and A498 cells come from clear cell RCC with VHL wild type [23 (link), 25 (link)], and clear cell RCC with VHL mutation (426_429delTGAC) [25 (link)], respectively. Cells were cultured in RPMI medium supplemented with 50 μg/mL of kanamycin and 10 % fetal bovine serum in an incubator at 5 % CO₂ and 37 °C. Human renal proximal tubular epithelial cell (HRPTEpC) was obtained from Cell applications Inc (San Diego, CA, USA). Cells were cultured in RenaEpi cell growth medium with growth supplements in an incubator at 5 % CO₂ and 37 °C. AR-A014418 was purchased from Calbiochem (San Diego, CA, USA). Two other GSK-3 inhibitors, SB-216763 and TDZD8, were obtained from Cayman Chemicals (Ann Arbor, MI, USA) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Rapamycin and everolimus were obtained from Selleck Chemicals (Houston, TX, USA), LY294002 was from Wako Pure Chemical Industries (Tokyo, Japan), recombinant GSK-3β was purchased from New England Biolabs (NEB) Japan (Tokyo, Japan), and recombinant GST-4EBP1 was obtained from Sigma-Aldrich Japan.