GST-CDK4 was purchased from Abcam, Inc (Cambridge, MA), while GST-CDK4/cyclin D1 was from Signal Chem, Inc (Richmond, Canada). For GST protein, E. coli. harboring GST or GST-CDK4 were grown overnight in room temperature. The recombinant proteins were induced with 1 mM IPTG for 4 hours. GST was purified by glutathione column and diluted to 1 ng/ul with 1X kinase assay buffer provided by the manufacturer (Cell Signaling, Inc, Danvers, MA). This was followed by combining 25 μl GST or GST-CDK4 with 1 ng substrate (GST-Rb) with or without 25 ng of cyclin D1, or HisTAG-MCM8, or HisTAG- ΔMCM8del261-290 or 2 ng of HisTAG-MCM8CBM in 1xkinase reaction buffer. The solutions were incubated at 37°C for 60 minutes. The reactions were terminated by adding 25 μl of 2N NaOH stop solution to each reaction well. The kinase activities were quantified using the kit and protocols of ADP-Glo™ Kinase Assay from Promega, Inc, Madison, WI.
For in vitro MCM8/cyclin D1/CDK4 binding assay, the following condition was used: 25 ng of each of HisTAG-MCM8, GST-cyclinD1 and GST-CDK4 produced from E.coli. were incubated with 150 mM NaCl, 25 mM MOPS, pH 7.2, 12.5 mM MgCl2, 12.5 mM β-glycerol phosphate at 37°C for 2 h. The products were then resolved in 6% non-denaturing polyacrylamide gel electrophoresis.
For in vitro MCM8/cyclin D1/CDK4 binding assay, the following condition was used: 25 ng of each of HisTAG-MCM8, GST-cyclinD1 and GST-CDK4 produced from E.coli. were incubated with 150 mM NaCl, 25 mM MOPS, pH 7.2, 12.5 mM MgCl2, 12.5 mM β-glycerol phosphate at 37°C for 2 h. The products were then resolved in 6% non-denaturing polyacrylamide gel electrophoresis.