A302 935

Cells were harvested with the lysis buffer previously described and centrifuged at 10,000× g at 4 °C, for 5 min. Protein content was determined by the Biorad Protein assay (Biorad). Proteins (20 μg–40 μg) were then subjected to SDS-PAGE under reducing and then transferred onto a nitrocellulose membrane. The rabbit anti-human WWP2 antibody (1/1000, A302-935, Bethyl Laboratories) and mouse anti-B actin antibody (1/1000, sc-8432, Santa Cruz Biotechnology) were used as primary antibodies and were detected using anti-rabbit or mouse IgG antibodies conjugated to horseradish peroxidase (1/10,000, Amersham Biosciences). Detection was done with the ECL chemiluminescence kit (Pierce) according to the manufacturer’s protocol.

Both p.R841 WWP2, p.R841H WWP2, and TRIF were expressed by using the TNT Quick Coupled Transcription Translation Systems kit (Promega), following manufacturer’s information. In vitro ubiquitination assay was determined with a ubiquitination kit (Enzo Life Science) according to manufacturer’s instructions. Briefly, E1 (100 nM) and UbcH5c (50 µg/ml) as an E2 were added for ubiquitination assays. The reactions were incubated for 90 min at 37 °C and stopped by adding 2x non-reducing gel loading buffer. Ubiquitin-conjugated TRIF was detected by immunoblot with an anti-ubiquitin antibody (1/1000, UBCJ2, ENZ-ABS840, ENZO). The expression levels of WWP2 were also verified by immunoblots with an anti-WWP2 antibody (1/1000, A302-935, Bethyl Laboratories).