VEEV capsid protein is ubiquitinated on K48.

<p>A) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 1, 2, 5 and 6 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 replicates in an experiment. Red boxes are parts of the image that have been zoomed in and displayed in Z-stacks. B) Number of infected U87MG cells showing co-localization tabulated. C) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were untreated (Mock), DMSO treated or Bortezomib treated (0.1μM) for 2 hours. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 2 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 independent experiments performed in duplicate. D) Number of infected U87MG cells showing co-localization tabulated. E) U87MG cells were treated with Bortezomib (0.1μM) or DMSO for 2 hours and then infected with TC-83 (MOI: 5) for 1 hour. At 2 hours post infection cell lysates were collected, lysed and quantified. Equal amounts of total protein were immunoprecipitated with capsid antibody and resolved by SDS-PAGE and subsequently immunoblotted for K48 ubiquitin (top panel) and capsid (bottom panel). The image is representative of 2 independent experiments. Protein bands were quantified using Image J software and normalized to capsid. Average percent differences of 2 independent experiments are depicted graphically.