Fig. 3.

NME1 localizes to the cortex of mitotic cells. (A) NME1 colocalizes with γ-tubulin in interphase and metaphase. HBE135 cells were grown on coverslips and fixed for immunofluorescence. Cells were stained with antibodies against NME1 (green) and γ-tubulin (red). DNA was stained with propidium iodide (red). Images of cells were captured as ≈50 0.37-μm confocal sections. middle, section through the middle of the cell; top, section at the top of the cell; projection, all sections projected onto a single image. (B) NME1 localizes to the cortex of mitotic cells. HBE135 cells were processed and stained with antibodies against NME1 (green). DNA was stained with propidium iodide (red). Confocal sections were obtained as in A. Images shown are merged images of a projection of all confocal sections and merged images of a section at the top of the cell. (C) NME1 cortical signal is absent in cells expressing the NME1 shRNA. HBE135 cells were transduced with lentiviral scrambled or NME1 shRNAs containing H2BGFP. After 32 h, cells were fixed and stained with anti-GFP and anti-NME1. Merged confocal projections are shown. shNME1, short hairpin NME1. (D) Flag-tagged NME1 localizes to the cortex. HBE135 cells were transduced with lentiviral Flag-tagged NME1. After 2 d, cells were fixed and stained with anti-Flag and anti-NME1 antibodies. Nuclei were stained with propidium iodide (PI). Confocal sections are as described in A. (Right) Zoomed images with arrowheads show colocalization (yellow) of the Flag (green) and NME1 (red) signals.