Tandem Affinity Purification of Ubiquitinated Proteins

TAP was performed as described previously, with minor modifications (Gloeckner et al., 2007 (link); Peters et al., 2013 (link)). RAW247.1 macrophages were infected overnight with 2 p.f.u. per cell and subsequently lysed in PBS supplemented with 0.5 % NP-40. Lysates were incubated with streptavidin beads (Thermo Scientific) for 2 h at 4 °C. Beads were washed three times and eluted in PBS supplemented with biotin (Thermo Scientific) for 1 h at 4 °C. Eluates were incubated with anti-FLAG M2 agarose beads (Sigma) and eluted in PBS supplemented with FLAG peptide (Sigma) in a similar manner. Final eluates were concentrated using 3 kDa Amicon filters (Millipore). Proteins retained on streptavidin and FLAG beads, as well as final eluates, were resolved in Novex 4–12 % Bis-Tris protein gels (Invitrogen) and analysed by silver staining (Invitrogen), or immunoblotting with anti-FLAG (Sigma-Aldrich) or anti-ubiquitin (FK2; Enzo Life Sciences) antibodies.