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

Santa Cruz sc-126) or 4 µg isotype mouse monoclonal antibodies (ThermoFisher Scientific #02-505 6200). Immunoprecipitated proteins were immunoblotted for GFP-or HA-epitope tagged 506 ubiquitin with antibodies for GFP (1:3,000, Roche #11814460001) or HA (1:4,000, Abcam 507 Cells were washed twice with room temperature PBS and harvested by scraping into 516 PBS, centrifuging at 100 x g for 4 minutes at 4˚C, and resuspending in PBS. Total cellular DNA 517 was purified using the Invitrogen PureLink DNA Purification Kit (Thermo Scientific #K1820-518 02) and quantitated by a NanoDrop Spectrophotometer. 10 ng of total cellular DNA was 519 analyzed by qPCR using custom primers specific to MAV-1 E1A (mE1Agenomic Fwd: 5' GCA 520 CTC CAT GGC AGG ATT CT 3' and mE1Agenomic Rev 5' GGT CGA AGC AGA CGG TTC 521 TTC 3') and the results were normalized to GAPDH, which was analyzed using a GAPDH-522 specific primer/probe set (ThermoFisher Scientific, Mm99999915_g1, #4331182). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 mRNA analysis by qPCR 526 Cells were harvested by scraping into media, centrifuging at 100 x g for 4 min at 4˚C, and 527 washing the cell pellet three times with ice-cold PBS. RNA was purified using the Qiagen 528