Key functional sites of SPINDLIN1 could be phosphorylated by Aurora-A. Key functional sites of SPINDLIN1 could be phosphorylated by Aurora-A. A, HeLa cells grown on coverslips were transfected with GFP-SPINDLIN1 and stained with Aurora-A antibody as well as DAPI. The different stages of the cell cycle from interphase to cytokinesis are shown. Scale bars, 10 μm. B, Coomassie-stained gels shows the prokaryotic expression and purification of recombinant GST and GST-SPINDLIN1 fusion proteins (top). The Western blotting shows the expression of GST-SPINDLIN1 and GFP-Aurora-A (middle). The proteins were mixed and incubated with glutathione beads, subjected to 10% SDS-PAGE, and immunoblotted (IB) with anti-Aurora-A antibody (input: 25 μg of protein extracts, bottom). C, HeLa cells were cotransfected with GFP-Aurora-A and C-MYC-SPINDLIN1 (left). Total cell lysates (500 μg) were used for immunoprecipitation (IP) with anti-C-MYC antibody or control mouse IgG. The immunocomplexes were analyzed by 10% SDS-PAGE and immunoblotted with anti-Aurora-A and anti-C-MYC antibodies (input: 25 μg of protein extracts, right). D, the full-length SPINDLIN1 sample was divided into 5 aliquots, and the purified GST-SPINDLIN1 fragments and full-length protein were incubated with immunoprecipitated Aurora-A in kinase buffer containing [γ32-P]ATP. The samples were analyzed by 10% SDS-PAGE and autoradiographed to determine the Aurora-A–specific phosphorylation region of SPINDLIN1 (top). In the selected region, SPINDLIN1 fragments were further divided into 4 aliquots and used in the in vitro kinase assay to narrow down the phosphorylation region (middle). Mutants of SPINDLIN1-S84A, S85A, S96A, and S99A were used to identify the phosphorylated sites by in vitro kinase assay (bottom). Jing-Xue Wang et al. Mol Cancer Res 2012;10:326-335 ©2012 by American Association for Cancer Research