Fig. 3.

Two distinct forms of pUb-IRAK1 identified by the combined use of Halo-NEMO and Halo-TAB2. (A) The experiment was carried out as in Fig. 2A except that pUb chains and associated proteins were captured with Halo-NEMO or Halo-TAB2 [first pull-down (PD)]. Where indicated, the supernatants from the first PD were subjected to a second PD using either Halo-NEMO or Halo-TAB2 as indicated. Captured pUb chains or IRAK1 were identified by immunoblotting. (B) As in A, except that proteins that were captured by Halo-NEMO or Halo-TAB2 from the supernatant of the first Halo-NEMO pull-down, were incubated with Otulin. The ubiquitylation of IRAK1 was then analyzed by immunoblotting with a specific antibody.