Fig. 2.

TNFα induces the formation of hybrid ubiquitin chains containing M1-Ub linkages. (A) HeLa cells, MEFs and THP1 monocytes were stimulated for 10 min with TNFα and ubiquitylated proteins captured from 2 mg cell extract protein and incubated with or without 1.0 μM Otulin as in Fig. 1. The samples were subjected to SDS/PAGE and immunoblotted with an antibody that recognizes all forms of RIP1. (B) As in A, except that human primary macrophages were stimulated with TNFα. (C) As in Fig. 1B, except that the samples were immunoblotted with the RIP1 antibody.