Streptavidin 680

The following antibodies were used in this study: ARS2 (XL14.1, 1:2000) generously provided by the Ludwig Institute for Cancer Research, Flag (1:1000, Cell signalling), Actin (1:4000, Sigma), TBP (1:1000, Cell Signalling), Tubulin (1:1000, Cell Signalling), GFP (1:1000, Cell Signalling), eRF1 (1:1000, Thermofisher), eIF3E (1:1000, Abclonal), eIF3F (1:1000, Abclonal), eIF3K (1:1000, Abclonal), Magoh (1:1000, Abclonal), MagohB (1:1000, Abclonal), eIF4A3 (1:1000, Abclonal), SMG7 (1:1000, Thermofisher), SMG1 (1:500, Santa Cruz), DHX34 (1:1000, Cedarlane), Streptavidin 680 (1:15000, Invitrogen), UPF1 (1:500, Santa Cruz), UPF1 (1:1000, Cell Signalling), NCBP1 (1:1000, Cell Signalling), Phospho-(Ser/Thr) ATM/ATR Substrate (1:1000, Cell Signalling).

Equivalent amounts of protein were boiled with 1x Laemmli buffer containing 50 mM DTT. Boiled protein samples were loaded for SDS-PAGE (Novex Wedgewell 4–20% Tris-glycine mini gels, Thermo XP04200BOX) and run at 120–140V for 90 min. Proteins were wet transferred to PVDF membranes, and membranes were blocked (Licor 927–60001) for 60 min. Tween (0.1%) and primary antibodies were added to membranes and incubated overnight at 4°C. Secondary antibodies were incubated with membranes in 50% Licor Odyssey blocking buffer/0.5x TBS/0.05% Tween/0.01% SDS at RT for 60 min. For visualization of biotinylated proteins, after incubation with primary antibodies, membranes were incubated in 50% Licor Odyssey blocking buffer/0.5x TBS/0.15% Tween/0.02% SDS. Streptavidin-680 (Invitrogen S21378, 1:5000) was incubated with membranes at room temperature for 60 min. Protein intensity was quantified with Licor Image Studio software.