Cdk6 cyclin d1

Cells’ extracts were subjected to immunoprecipitation using the anti-NPM/B23 antibody. Immunocomplexes were washed with WCE buffer and kinase reaction buffer (25 mM Tris (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, and 10 mM MgCl₂). One μg of CDK6/Cyclin D1 (Invitrogen) was added to the immunocomplexes for 1 h at 30 °C. For inhibition studies, immunocomplexes and CDK6/Cyclin D1 were incubated in the presence of 2 μM palbociclib. The degree of phosphorylation was analyzed with both SDS-PAGE and immunoblotting.

Titanium dioxide (titania) particle (10 μm diameter) were purchased from GL Sciences (Tokyo, Japan). Thermo-sensitive alkaline phosphatase (TSAP) and modified trypsin were obtained from Promega (Madison, WI). [²H₂, ¹³C]Formaldehyde was purchased from ISOTEC (Miamisburg, OH). Recombinant human protein kinases were from Carna Biosciences (Kobe, Japan), with the exception of KHS1, SGK496, NIK, CDK6/Cyclin D1, CDK9/Cyclin K, ALK1, CDK4/Cyclin D1, BARK1 and MPSK1 (Invitrogen, Carlsbad, CA) and GPRK5, FRAP, DAPK2, PKG1a, STK33, TAO1 and ULT3 (Millipore, Billerica, MA). More details on kinases are shown in Supplementary Table S5 and Supplementary Information. All other reagents were from WAKO Chemicals (Osaka, Japan).

Inhibition of the catalytic activities of protein kinases, CDK4–cyclin D1 and CDK6–cyclin D1 (Invitrogen, Carlsbad, CA, USA), by GLR2007 (Gan & Lee Pharmaceuticals, Beijing, China) and palbociclib (Selleck Chemicals LLC, Houston, TX, USA) was assessed using a LANCE Ultra time-resolved fluorescence resonance energy transfer assay (PerkinElmer Inc., Waltham, MA, USA) according to the manufacturer’s instructions. GLR2007 was tested across a range of concentrations from 0.0017 nM to 100 nM. palbociclib was tested across a range of concentrations from 0.0169 nM to 1000 nM. Results were calculated as half-maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i). IC₅₀ and K_i values were determined using the XLfit 205 model (ID Business Solutions Ltd, Guildford, UK) curve-fitting add-on for Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).