Fig. 2

BMI1 is a substrate of CK2α. a Co-precipitation of BMI1 with CK2α. Reciprocal immunoprecipitation (IP) assay was performed with BMI1 and CK2α antibodies crosslinked with the agarose resin and immunoprobed with CK2α and BMI1 antibodies respectively. b In vitro kinase assay with CK2α and BMI1. In vitro kinase assay was performed with 400nM CK2α, 200nM or 400nM BMI1-GST and radioactive ATP and representative autoradiograph image is presented.. Reaction mixture without substrate (lane 1), only GST protein (lane2), without enzyme (lane 3), and enzyme with GST only (lane 4) served as negative controls. c Kinase assay with immunoprecipitated (IP) endogenous CK2α and purified BMI1. CK2α was immunoprecipitated from CP20 cells using agarose A/G beads, the beads was washed and incubated in a kinase assay buffer supplemented with purified BMI1-GST and radiolabeled ATP, in presence or absence of the specific CK2 inhibitor TBB. A representative autoradiograph is provided in the right panel. Efficient Immunoprecipitation is demonstrated in the left panel by immunoblotting a small fraction of the IPed beads with CK2α antibody