2.7. Immunoprecipitation

Two milligrams of total protein were incubated overnight at 4 °C with rotation using 2 µg mouse IgG3 isotype control (Abcam, 18394, Cambridge, UK) or anti-VEEV nsP2 antibody (KeraFast, EU015) or anti-MATRN3 (Novus, NB100-1761). Magnetic Dynabeads coated with protein G (FisherSci, 10-003-D) were washed with citrate phosphate buffer pH 5.0 (50 mM Tris-HCL pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na₃VO₄, Protease cocktail tablet) (Sigma-Aldrich, 11697498001, St. Louis, MO, USA) and added to the protein–antibody IP complexes. Rotation at room temperature proceeded for 40 min followed by 1× wash with TNE100 + 0.1% NP-40, 1 wash with TNE50 + 0.1% NP-40, and 2 wash with PBS. TNE buffers consisted of 100 mM Tris-HCl pH 7.5 and 0.2 mM EDTA, with 100 mM NaCl for TNE100 or 50 mM NaCl for TNE50. For Western blot imaging, Laemmli buffer supplemented with 100 mM DTT was added and beads were boiled for 10 min. For samples subjected to mass spectrometry, the last PBS wash was removed and Dynabeads were stored at 80 °C until processed for analysis.