Molecules and Cells

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Fig. 1.

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Fig. 1. (A) The DDA3 complex was purified from mitotic cells and analyzed by mass spectrometry. A peptide of c-Cbl was identified three times. (B) Twenty-eight hours after transfection of the EV or HA-c-Cbl plasmid, HeLa cells were harvested and subjected to immunoprecipitation and western blotting with the indicated antibodies. p38MAPK served as a loading control. EV, empty vector. (C and D) HeLa cells were synchronized by a double thymidine block (C) or thymidine-nocodazole block (D), placed into fresh media, and harvested at the indicated times. Cell lysates were analyzed by western blotting with the indicated antibodies. AS, unsynchronized cells. (E) HeLa cells were transfected with control (siControl) or c-Cbl-specific siRNAs (siCbl-A and siCbl-B). Seventy-two hours after siRNA transfection, the transfected cells were harvested and lysed to measure protein levels by western blotting with the indicated antibodies. (F and G) Seventy-two hours after siRNA transfection, HeLa cells were fixed with MeOH and stained with antibodies as indicated. Images are maximum projections from Z-stacks of representative cells stained for c-Cbl (green), β-tubulin (red), and DNA (blue). The number of metaphase cells with unaligned chromosomes was quantified and plotted (G) (n = 300 metaphase cells from three independent experiments). (H) Seventy-two hours after siRNA transfection, HeLa cells expressing GFP-Histone H2B cells were imaged for GFP fluorescence by time lapse. Images were captured every 3 min to monitor mitotic progression. NEB, nuclear envelop breakdown. Unaligned, the initial formation of the metaphase plate. Data are represented as mean ± SEM. Scale bars = 5 μm. *P < 0.01.
Mol. Cells 2019;42:840~849 https://doi.org/10.14348/molcells.2019.0142
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