(A,B) RAW264.7 cells were stimulated for 20 min (A) or 6 h (B) with the TLR7-activating ligand R848 (250 ng/ml) and ubiquitylated proteins were captured from the cell extracts on Halo-NEMO beads. The Halo-NEMO pull downs were incubated for 1 h without (−) or with (+) 0.5 M hydroxylamine at pH 9.0 followed by incubation without (−) or with (+) the broad specificity deubiquitylase USP2 (1 μM). (C,D) The ubiquitylated proteins captured from the extracts of BMDM from wild-type (WT) or HOIL-1[C458S] mice (C458S) were incubated for 1 h without (−) or with (+) 0.5 M hydroxylamine at pH 9.0 and analysed as in (A,B). (E,F) As in (A B) except that, prior to SDS–PAGE and immunoblotting, the Halo-NEMO resin was incubated with 1 µM Otulin (lanes 5 and 6), 1 µM Otulin plus 1 µM AMSH-LP (lanes 7 and 8) and Otulin plus AMSH-LP and then 0.5 M hydroxylamine (lanes 9 and 10), or 1 µM USP2 alone (lanes 11 and 12) (see Methods).