Rabbit anti xrn1

Cells were washed and pelleted in cold PBS, followed by resuspension in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1mM EDTA, 0.5% NP-40, and protease inhibitor (Roche)). Cell lysates rotated for 1 h at 4°C and were clarified by centrifugation at 21,000 x g for 10 min. 10–20 μg whole cell lysate were used for SDS-PAGE. Western blotting was done in Tris-buffered saline and 0.1% Tween 20 (TBS-T) using the following primary antibodies: rabbit anti-ORF59 (1:10,000), rabbit anti-K8.1 (1:10,000), rabbit anti-vinculin (1:1,000; Abcam), streptavidin-HRP (1:500 Thermo Fisher), rabbit anti-XRN1 (1:1,000; Bethyl), mouse anti-CASK (1:1,000; Abcam), rabbit anti-PABPC1 (1:1,000; Cell Signaling Technologies), rabbit anti-ORF6 (1:10,000), rabbit anti-PABPC4 (1:1,000; Bethyl), mouse anti-ORF26(1:500; Novus), rabbit anti-ORF68 (1:5,000), mouse anti-GAPDH (1:1,000; Abcam), rabbit anti-ORF29 (1:500). Rabbit anti-ORF59, K8.1, ORF6, and ORF68 have all been previously described [8 (link), 54 (link)]. Preparation of the rabbit anti-ORF29 antibody is described below.