Xenbase Image ID: 118870

Figure 4. Determination of Aurora A phosphorylation sites of TACC3 in vitro. (A) Consensus sequences for Aurora A phosphorylation in Xenopus TACC3/maskin. Yellow boxes indicate conserved domains among the TACC family, whereas green boxes are the domains that are highly conserved with TACC3 homologues only. The 3A mutant protein (TACC3-3A) has mutations of alanine substitution on three serine residues (Ser33, Ser620, and Ser626) in the consensus sequences of Aurora A phosphorylation. Numbers represent the positions for amino acid residues of Aurora A phosphorylation target sites in the amino acid sequence of TACC3. (B) Aurora A kinase assay with recombinant WT versus alanine mutant TACC3 proteins. WT or 3A mutant TACC3 was incubated with or without recombinant Aurora A (−/+ Aurora A) in the presence of γ−[32P] ATP (see Materials and methods). The reaction mixture was loaded onto SDS-PAGE, and the gel was stained with Coomassie brilliant blue (CBB; lanes 1–4). The incorporation of 32P into TACC3 in the gel was measured by autoradiography (32P; lanes 5–8). (C) Characterization of phosphospecific antibodies. TACC3-WT was incubated with or without Aurora A (−/+ Aurora A), and the reaction mixture was loaded onto SDS-PAGE. The blots were probed with anti-TACC3 antibody (lanes 1 and 2), antiphospho-Ser33 (lanes 3 and 4), antiphospho-Ser620 (lanes 5 and 6), and antiphospho-Ser626 (lanes 7 and 8). Image published in: Kinoshita K et al. (2005) Copyright © 2005, The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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