The influenza A virus endoribonuclease PA-X usurps host mRNA processing machinery to limit host gene expression

Lea Gaucherand; Brittany K. Porter; Summer K. Schmaling; Christopher Harley Rycroft; Yuzo Kevorkian; Craig McCormick; Denys A. Khaperskyy; Marta Maria Gaglia

Neutravidin pull-down 546 60 µl of 50% slurry of High Capacity Neutravidin Agarose Beads (Thermo) was used for each 547 500 µl of clarified whole cell lysate. Beads were equilibrated in RIPA buffer by washing three 548 times for 10 mins at 4°C. In one of the BirA*-X61 experimental runs, 1 µl of 500x RNase A 549 (100 µg, Qiagen) was added to each sample to remove non-specific interactors. The lysate was 550 then incubated for 5 minutes at room temperature before loading onto the beads. Untreated 551 samples were loaded directly onto the beads post washing. 1 mg of protein sample was loaded to 552 beads in 1.5 mL Eppendorf tubes, which were then placed on a rotator overnight at 4°C, and 553 collect with centrifugation at 400 x g for 1 minute at 4°C. Beads were washed with RIPA buffer 554 three times, followed by three washes with TAP buffer (50 mM HEPES-KOH pH 8.0, 100 mM 555