Ubiquitinylation kit bml uw9920

The in vitro ubiquitination assays were performed with an Ubiquitinylation kit (BML-UW9920, Enzo Life Sciences), using either UbcH6 or UbcH5b E2 enzymes and following the manufacturer’s protocol. For LjFLS2 ubiquitination tests UbcH5c E2 enzyme was also used.
All the reactions were incubated at 30 °C for 3 h, and then stopped by adding SDS sample buffer and boiled at 98 °C for 5 min. The samples were then separated by SDS–PAGE and analyzed by Western blotting, using the a-GST antibody (GE Healthcare) and the a-HIS antibody (Roche) to detect the tagged proteins or the anti-ubiquitin antibody (Santa Cruz Biotechnology P4D1) to detect the ubiquitinated fraction.

In cellulo ubiquitination was performed as previously described^{30 (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.