Fig. 4: Aurora B kinase phosphorylates caspase-2 at S384 in vitro.
From: Phosphorylation by Aurora B kinase regulates caspase-2 activity and function
a, b In vitro interaction between purified recombinant GST-Casp2-C320G (GST-Casp2-fl) or GST-Casp2363–423-WT and His-AURKB was determined by GST pull-down assay. Stain-free membrane shows protein loading amount for GST or GST-Casp2. Antibodies used are indicated. c U2OS-CASP2−/− cells stably expressing GFP (sKO) or GFP-caspase-2-C320G (s320G) were used. GFP or GFP-caspase-2-C320G were immunoprecipitated (IP) with GFP-Trap from the asynchronous (Async), mitotic or ZM447439 (ZM)-treated cells and subjected to immunoblotting with the indicated antibodies. Phospho-histone H3 (pHis-H3) was used as mitosis marker. Stain-free membrane was used as loading control. d GST-Casp2-fl WT, GST-Casp2-fl S384A, GST-Casp2363–423 WT, GST-Casp2363–423 S384A or GST was subjected to in vitro phosphorylation by incubation with AURKB and [γ-32P]-ATP and analysed by autoradiography. IB with GST antibody shows equal loading. MBP, myeloid basic protein was used as positive control for AURKB phosphorylation.
