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

Sample Buffer (Invitrogen Cat #NP0007), and 1.25 µL 1M DTT. Samples were incubated at 473 37˚C for 10 minutes and then loaded into a well of an 8% acrylamide gel (8.3 cm wide x 7.3 cm 474 high x 0.1 cm thick) with a 2.5% stacking gel, electrophoresed for 30 minutes at 50 V and 85 475 minutes at 150 V, and then transferred to a PVDF membrane (BioRad #1620177) for 1 hour at 476 100V at 4˚C. Blots were blocked in 5% bovine serum albumin (BSA, Sigma A7906) in tris-477 buffered saline (BioRad #1706435) and 0.1% Tween 20 (Sigma P1379). Blots were probed with 478 primary antibodies to detect mouse PKR (Santa Cruz D-20 sc-708, 1:2000, or B-10 sc-6282, 479 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 1:200), mouse actin (Santa Cruz sc-1616-R, 1:1000), MAV-1 E1A (AKO-7-147, 1:1000, 480 described previously (68)), or MAV-1 late viral proteins (AKO 1-103, 1:1000, described 481 previously (91, 92)). Secondary antibodies used were IRDye 800CW anti-rabbit (Li-Cor 925-482 32213, 1:15,000) or IgG peroxidase-conjugated anti-mouse (Jackson Immuno 515-035-062, 483 1:20,000). Blots were visualized by LI-COR Odyssey imaging (LI-COR Biosciences) or 484 enhanced chemiluminescent substrates (Pierce ECL Western Blotting Substrate #32106) and X-485 ray film (Dot Scientific #BDB810). Densitometric quantification was performed on .tif files 486 using ImageJ software from NIH (93). 487