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. 2015 Oct 22;6(10):e1943. doi: 10.1038/cddis.2015.295

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Copyright © 2015 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Effect of Pur-alpha and FUSMM expression on protein synthesis. (a) Untransfected HeLa cells or cells transfected with HA-Pur-alpha, Flag-FUSWT, or Flag-FUSMM were treated with puromycin. Untransfected cells are shown in the first row, while cells transfected with HA-Pur-alpha are in the second row. Cells with higher expression of Pur-alpha (transfected cells) show no incorporation of puromycin, compared with untransfected cells of the same field. Puromycin incorporation was detected by immunofluorescence with anti-puromycin antibody. Cells were also labeled by immunofluorescence with anti-Pur-alpha and anti-Flag antibodies (bars=10 μm). (b) Cytoplasmic extract from HEK293 cells was separated by ultracentrifugation on a linear sucrose gradient. Eleven fractions were collected monitoring the absorbance at 260 nm and the pellet containing polyribosomes was pooled to the first fraction. The upper panel shows the absorbance profile with the position of ribosomal subunits and monomer indicated. Proteins purified from the fractions were analyzed by western blot with the indicated antibodies