Anti pabpc1

Protein concentration of cell lysates was determined with Pierce Coomassie (Bradford) Protein Assay kit (Thermo Fisher Scientific, 23200). Proteins were analyzed by SDS-Page (Thermo Fisher Scientific, EA0375BOX) using Westran Clear signal PVDF membranes (Sigma Aldrich, 0485289) and the following antibodies: anti-HA.11 epitope tag antibody (Covance, MMS-101P), anti-DDX3X (Bethyl Laboratories, A300-474A), anti-PABPC1 (Cell Signaling, 4992), anti-TRIM21 (New England BioLabs, 92043) and anti-V5 Clone 5C5 (gift from M. Busslinger). The protein size was determined with a Spectra Multicolor High Range Protein Ladder (Thermo Fisher Scientific, 26625). Signal was detected with Pierce ECL Western blotting substrate (Thermo Fisher Scientific, 32209), or Amersham ECL select Western blotting detection reagent (GE Healthcare Life Sciences, RPN2235). Visualization was performed with the chemiluminescent gel documentation system MF-chemi 3.2 Pro (DNR Bio-Imaging Systems) respectively with the Bio-Rad ChemiDoc XRS Gel Documentation system (Bio-Rad Laboratories).

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).