GTSE1 protein expression peaks during G2 and M phase and localizes to mitotic spindle microtubules. (A) Western blot showing GTSE1 protein expression from HeLa cells after release from nocodazole for the indicated times. The N lane indicates cells in nocodazole and the 0 time point. The G2 lane indicates the adherent cell population that was collected after mitotic shakeoff from the 0 time point (N lane). The Thy lane indicates cells that were released from nocodazole after 16 h directly into thymidine for 24 h. Cyclin B protein levels were also probed to show cell cycle specificity. Actin is shown as a control for loading levels. Cells exit mitosis by ∼4 h after release from nocodazole as denoted by cyclin B degradation. GTSE1 levels remained very low after exit from mitosis until ∼20 h after release from nocodazole, which is a time consistent with reentry into G2 phase. During thymidine-induced S phase arrest (Thy lane), GTSE1 protein accumulated. (B) Immunofluorescence images of endogenous GTSE1 in unsynchronized HeLa cells during interphase and different stages of mitosis. GTSE1 was largely undetectable in most interphase cells. During late G2/prophase, it began to accumulate near centrosomes and became concentrated on mitotic spindle microtubules during mitosis. Cells were coimmunostained for DNA (DAPI) and α-tubulin. Images represent maximum-intensity projections. GTSE1 images are scaled equivalently. Bar, 10 µm.