TPX2 mutants abolish the in vivo interaction between TPX2 and Aurora A as well as Aurora A localization to spindles. (a) Western blots of cell extracts immunoprecipitated to detect interaction of TPX2 with Aurora A. U2OS (untagged), mTPX2^WT, mTPX2^AAA, and mTPX2^ΔN cells were arrested in mitosis with nocodazole, and protein extracts were immunoprecipitated with anti-GFP antibody. Input and immunoprecipitated fractions were run by SDS-PAGE and blotted with either anti-mTPX2 or anti–Aurora A antibody. Anti-GFP antibody pulls down mTPX2^WT bound to Aurora A, whereas nothing is immunoprecipitated in the untagged cells. Immunoprecipitating mTPX2^WT does not pull down endogenous TPX2. In mTPX2^AAA and mTPX2^ΔN cells, the mTPX2 mutant transgene is pulled down, but Aurora A is not. The single asterisk represents the position of the TPX2^ΔN protein. The double asterisk represents a degradation product of the mTPX2 protein. (b) Immunofluorescence analysis of cell lines after CON or TPX2 RNAi stained for α-tubulin (green), DNA (blue), mTPX2-LAP (anti-GFP; insets), and Aurora A. Aurora A is localized to spindles and centrosomes in U2OS cells (top row). After hTPX2 RNAi, Aurora A is absent from spindles but still on centrosomes (second row). A mouse TPX2-LAP transgene restores Aurora A localization to spindles after hTPX2 RNAi (third row). The same mouse transgene with point mutations introduced to abolish the TPX2–Aurora A interaction no longer is able to recruit Aurora A to spindles after hTPX2 RNAi (bottom row). The pericentrosomal Aurora A localization shown in the mutant similar to that of the TPX2 depletion was observed in images of 12 of 14 cells. In 2 of 14 images, the Aurora A localization in the mutant reflected a more centrosomal localization. Bar, 10 μm.