In cellulo ubiquitination was performed as previously described30 (link). HEK293T cells were transiently transfected with pIRES-HuR, pcDNA-6His-Ubiquitin, and pcDNA-Pirh2 vectors. An empty pcDNA3.1 plasmid was used as negative control. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 16 h and then lysed in 8 M urea-containing buffer (pH 8.0). 6His-ubiquitinated proteins were purified on Ni-NTA beads (Qiagen, Hilden, Germany). The bound proteins were eluted and analyzed by western blotting.
In vitro ubiquitination assay was performed using Ubiquitinylation kit (BML-UW9920; Enzo Biochem, New York, NY, USA) according to the manufacturer’s recommendations. The purified GST-tagged proteins were used for in vitro ubiquitination reaction: HuR-GST was used as a substrate; full-length Pirh2 (Pirh2 FL-GST), N-terminal domain of Pirh2 (Pirh2 NTD-GST), and GST as the negative control were used for their E3 in vitro ligase activity investigation. The protein ratios used for the reaction are demonstrated in Supplementary Fig.S5 .
In vitro ubiquitination assay was performed using Ubiquitinylation kit (BML-UW9920; Enzo Biochem, New York, NY, USA) according to the manufacturer’s recommendations. The purified GST-tagged proteins were used for in vitro ubiquitination reaction: HuR-GST was used as a substrate; full-length Pirh2 (Pirh2 FL-GST), N-terminal domain of Pirh2 (Pirh2 NTD-GST), and GST as the negative control were used for their E3 in vitro ligase activity investigation. The protein ratios used for the reaction are demonstrated in Supplementary Fig.
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