Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Danielle E. Goodman; Carla D. Pretto; Tomas A. Krepostman; Kelly E. Carnahan; Katherine R. Spindler

To attempt to demonstrate PKR ubiquitination status during MAV-1 infection, C57BL/6 489 MEFs were transfected with GFP-or HA-epitope tagged ubiquitin plasmids (Addgene #11928 490 and #18712, respectively) 24 hours before infection. We used Polyplus jetPRIME transfection 491 reagent (Polyplus #114-15) with 10 µg plasmid DNA and 30 µL jetPRIME reagent per 10 cm 492 plate. At 12 hpi (36 hours post transfection), we treated mock and infected C57BL/6 MEFs with 493 10 µM MG132 (Sigma M7449) for 6 hours before collecting lysates at 18 hpi in HCN buffer (50 494 mM HEPES, 150 mM NaCl, 2 mM CaCl 2, 1% Triton X-100 (Sigma T9284), 1x protease 495 inhibitors (Protease Inhibitor Cocktail Kit, Thermo Scientific #78410), and 5 mM N-496 ethylmalemide). The lysates were split into two aliquots, and 3 µg PKR (D-20 sc-708, 497 discontinued) or 3 µg isotype rabbit polyclonal antibody (Jackson Immuno #011-000-002) was 498 added to lysates. After rocking samples overnight at 4˚C, 20 µL protein A agarose suspension 499 (Calbiochem/Millipore #IP02-1.5ML) was added to each and samples were rocked at 4˚C for 2 500 hours. After incubation, agarose was washed 3 times with 1 mL HCN buffer, resuspended in 40 501 µL 2x Laemmli buffer (BioRad #161-0737) with 5% 2-mercaptoethanol (Sigma M6250), and 502 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/584680 doi: bioRxiv preprint boiled for 10 minutes. Lysate supernatants remaining after the initial PKR immunoprecipitation 503 were then immunoprecipitated again using the same procedure but with 4 µg anti-p53 (DO-1, 504