Molecules and Cells

Indexed in /covered by CAS, KoreaScience & DOI/Crossref:eISSN 0219-1032   pISSN 1016-8478

Fig. 2.

Download original image
Fig. 2. (A) Forty-eight hours after siRNA transfection, HeLa cells were transfected with HA-Ubiquitin plasmid. Twenty-four hours after plasmid transfection, the cells were treated with 10 mM proteasome inhibitor, MG132, for 5 h and then harvested and subjected to immunoprecipitation and western blotting with the indicated antibodies. (B) Seventy-two hours after siRNA transfection, the cells were treated with nocodazole for 18 h. The mRNA levels of c-Cbl and DDA3 in mitotic cells were quantified by real-time RT-qPCR analysis (n = 3). (C and D) Seventy-two hours after siRNA transfection, HeLa cells were harvested to analyze protein levels with the indicated antibodies (C) or fixed with MeOH to analyze unaligned chromosomes in metaphase cells (D). Relative intensities of band were quantified by image processing software (C; Image Studio ver5.0). (E) Seventy-two hours after siRNA transfection, HeLa cells were fixed with MeOH and stained with antibodies as indicated. Images, which were acquired under a constant exposure time, are maximum projections from Z-stacks of representative cells. The intensity of DDA3 was quantified and plotted (n = 10 cells for each quantification). (F) HeLa cells were transfected with siRNA and prepared as in . For each quantification, the intensities of spindle and Kif2a were determined based on the immunofluorescence from 10 cells. (G) Forty-eight hours after siRNA transfection, HeLa cells were transfected with either the EV or HA-c-Cbl plasmid. The cells were fixed in MeOH and stained with β-tubulin antibody at 28 h after plasmid transfection. Images for β-tubulin were acquired under a constant exposure time and spindle intensity was quantified (n = 10 cells for each quantification). EV, empty vector; AU, arbitrary units. 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
© Mol. Cells