Anti xrn1

Cells transfected for 3 days were washed with PBS once and lysed in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) in the presence of cOmplete™, EDTA-free protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were clarified to remove the non-soluble fraction by centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentrations were measured by Bradford Assay and 50 μg of total protein lysate was mixed with 5X protein sample buffer containing reducing agent. Samples were separated on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore), and blocked with 5% non-fat milk in PBS-T. The following primary antibodies were used to probe the membranes: anti-Core (C7-50) (Abcam, ab2740), anti-Actin (Sigma), and anti-XRN1 (Bethyl Lab A300-443A.) Immunoblots were developed using Pierce ECL Western Blot Substrate (Thermo Fisher) following the manufacturer’s suggested instructions.

Protein extraction was performed with peqGOLD TriFast reagent (VWR), and proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (GE Healthcare Life Sciences). The following antibodies were used: anti-CASC3 amino acid residues 653–703 (Bethyl Laboratories, #A302-472A-M), anti-CASC3 amino acid residues 367–470 (Atlas Antibodies, #HPA024592), anti-EIF4A3 (Genscript), anti-FLAG (Cell Signaling Technology, #14793), anti-MAGOH (Santa Cruz Biotechnology, #sc-271365), anti-PABPC1 (Cell Signaling Technology, #4992), anti-RBM8A (Atlas Antibodies, #HPA018403), anti-SMG6 (Abcam, #ab87539), anti-SMG7 (Elabscience, #E-AB-32926), anti-Tubulin (Sigma-Aldrich, #T6074), anti-UPF3B (antiserum was raised in rabbits by Eurogentech against a C-terminal fragment of UPF3B (300–483) and affinity purified), anti-V5 (QED Bioscience, #18870), anti-XRN1 (Bethyl Laboratories, #A300-443A), anti-rabbit-HRP (Jackson ImmunoResearch, #111-035-006), anti-mouse-HRP (Jackson ImmunoResearch, #115-035-003). Detection was performed with Western Lightning Plus-ECL (PerkinElmer) or Amersham ECL prime (GE Healthcare Life Sciences) and the chemiluminescence imager Fusion FX6 EDGE (Vilber-Lourmat).