Author: Nila Roy Choudhury; Gregory Heikel; Maryia Trubitsyna; Peter Kubik; Jakub Stanislaw Nowak; Shaun Webb; Sander Granneman; Christos Spanos; Juri Rappsilber; Alfredo Castello; Gracjan Michlewski
Title: RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination Document date: 2017_10_9
ID: ifla4aix_83
Snippet: Immunoprecipitation was performed as described above. Following the final wash proteins bound to beads were incubated with Ube1, His-Ube2D3 and ubiquitin (UBPBio). 1 mg/ml Ube1, 2 mg/ml His-Ube2D3 and 50 mg/ml ubiquitin was used for TRIM25 autoubiquitination. 5 mg/ml Ube1, 10 mg/ml His-Ube2D3 and 250 mg/ml ubiquitin was used for ZAP ubiquitination. The reactions were carried out at 37C for 1 hour or as indicated and were stopped by addition of lo.....
Document: Immunoprecipitation was performed as described above. Following the final wash proteins bound to beads were incubated with Ube1, His-Ube2D3 and ubiquitin (UBPBio). 1 mg/ml Ube1, 2 mg/ml His-Ube2D3 and 50 mg/ml ubiquitin was used for TRIM25 autoubiquitination. 5 mg/ml Ube1, 10 mg/ml His-Ube2D3 and 250 mg/ml ubiquitin was used for ZAP ubiquitination. The reactions were carried out at 37C for 1 hour or as indicated and were stopped by addition of loading buffer and reducing agent. The protein ubiquitination was analysed by Western blot with anti-TRIM25 or anti-ZAP antibodies.
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