Mps1 bears a putative nuclear export sequence. (A) Prediction of nuclear export sequence in Mps1 via 2 online procedures. Mps1 protein sequence was loaded into procedures according to the protocol by software developer. The resulted nuclear sequences were showed as indicated. (B, C) Functional validation of the putative NES. The putative sequences were fused with EGFP in frame and the resulted constructs were transfected into 293 T cells. The subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. DNA was counterstained with PI. The ratio of fusion proteins in the nucleus and cytoplasm were measured via image J and the statistical result of these were generated by using Graphpad software. (D) The amino acids alignment of NES motifs. The pNES1 of Mps1 is aligned with the classical NES from MAPKK, Cyclin B1, HIV-REV. The letters in red indicate identical or similar amino acids. PI, Propidium Iodide.