Assaying C/EBPβ Ubiquitination In Vivo and In Vitro

In vivo and in vitro ubiquitination assay were performed as previously described [60 (link)]. For C/EBPβ in vivo ubiquitination assay, the BV2 cells or 293T cells that transfected with the indicated expression vectors were treated with MG132 for 4 hours and then were lysed with lysis buffer containing protease inhibitors and N-ethylmaleimide (Sigma). The cell extracts were boiled for 5 minutes in the presence of 1% SDS to dissociate the C/EBPβ-interacting proteins and then were diluted with lysis buffer until the concentration of SDS was 0.1% before immunoprecipitation. C/EBPβ was then immunoprecipitated from the cell extracts and was assessed the status of ubiquitination with anti-ubiquitin antibody. For C/EBPβ in vitro ubiquitination assay, the HA-Peli1, HA-Peli1ΔC, and C/EBPβ were translated in vitro by using cell-free quick coupled transcription/translation system (L1170, Promega). The ubiquitination reaction was processed by mixing the translated proteins with precharged ubiquitin-conjugating enzyme E2 UbcH5a (E2-800, Boston Biochem) and incubating at 37°C for 4 hours according to the manufacturer’s protocol. The reaction was then terminated by boiling for 5 minutes in SDS loading buffer, and the samples were subjected to SDS-PAGE and immunoblotting to detect the ubiquitination of C/EBPβ mediated by Peli1.