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. 2018 Oct 25;293(52):20062–20072. doi: 10.1074/jbc.RA118.005449

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© 2018 Tang et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 4. — M1-linked ubiquitination of cFLIP inhibits Lys-48-linked ubiquitination. A, LUBAC-induced in vitro ubiquitination of cFLIP. The recombinant FLAG-cFLIP was incubated as indicated and probed with anti-cFLIP antibody. E1 is UBE1, and E2 is UbcH5c. Long Ex, long exposure time; Short Ex, short exposure time. B, Western blot analysis of immunoprecipitates (IP) from 293T cells transiently transfected with the indicated constructs using anti-FLAG beads. C, Western blot analysis of immunoprecipitates from lysates of control and RNF31-silenced HeLa cells stably expressing FLAG-cFLIP after pretreatment with MG132 followed by treatment with TNFα and CHX (20 ng/ml and 10 μg/ml, respectively) for 1 h using anti-FLAG beads.