B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody. 