Fig. 4

RIP1 ligase has autoubiquitination activity and inhibits degradation of RMD. a MBP-labelled RIP1 was assayed for ubiquitination activity in the presence (+) or absence (−) of E1 (from wheat), E2 (AtUBC8) and 6 × his-tagged ubiquitin (Ubi) using anti-ubiquitin antibody. A plain MBP tag was used in the negative control lacking RIP1. b, c, e RMD protein extract was mixed with different ratios (0, 1, 2, 4, or 8 × vol) of RIP1-eGFP extract. The sixth lane contains a 9 × vol of RIP1 extract with no RMD extract. Anti-RMD antibody (Ab) was used to detect RMD; anti-eGFP antibody was used to detect tagged RIP1. Protein amounts: b immediately after mixing (0 min); c 10 min after mixing; e 10 min after mixing in the presence of 50 μM MG132, a proteasome inhibitor. d, f Quantification of western blot result from (c) and (e), respectively. a.u., arbitrary unit