Prk5 ha ubiquitin wt

pSG5-mouse Rxra (Leid et al., 1992 (link)) and pCMV6-mouse Rnf8 (Origene) expression plasmids were co-transfected into Hepa-1c1c7 cells (ATCC) using Lipofectamine 3000 (Thermo Fisher Scientific). pRK5-HA-Ubiquitin-WT (Addgene) were co-transfected for polyubiquitin type analysis. Three days after transfection, cell lysates were harvested with RIPA buffer. For ubiquitination assays, 20 μM MG-132 (Sigma-Aldrich) was added to the medium and the cells were cultured for 4 h before harvesting. For RXRα immunoprecipitation, rabbit monoclonal RXRα antibody (Abcam) was added to 400 μL cell lysate adjusted at 1 mg/ml protein concentration. The cell lysate was incubated at 4°C for overnight. The following day, protein A agarose (Sigma-Aldrich) was added and incubated for 2 h at 4°C. Then, the RXRα-ubiquitin complexes were added to Laemmli sample buffer (Bio-Rad) with 5% 2-mercaptoethanol and heated to 95°C for 5 min. The samples were subsequently analyzed by western blot.

All experiments using expression of BCLb proteins were performed with vectors engineered for this study. The following accession numbers were used to design oligos for PCR based cloning from cDNA libraries generated from a “universal human RNA” library (Stratagene/Agilent Technologies): BCLb #NM_020396. FLAG-tagged BCL2 constructs were generated by performing PCR of the MIG-BCL constructs using an oligo that fused the FLAG coding sequences to the amino terminus of each BCL2-protein. This product was then subcloned back into the MIGRX vector. The following plasmids were obtained from Addgene; FLAG-Ubqln (Addgene plasmid 8663, deposited by Peter Howley), FLAG-Ubqln-112X (Addgene plasmid 8664, deposited by Peter Howley), pRK5-HA-Ubiquitin-WT (Addgene plasmid 17608, deposited by Ted Dawson), pRK5-HA-Ubiquitin-K0 (Addgene plasmid 17603, deposited by Ted Dawson), pRK5-HA-Ubiquitin-K48 (Addgene plasmid 17605, deposited by Ted Dawson). Lysine-less BCLb construct was engineered using the MIG- or MIT-BCLb constructs described above to do site directed mutagenesis with the Quick Change Multi Site Mutagenesis kit (Stratagene/Ambion), as per the manufacturer’s protocol. MYC-UBQLN1 construct was a gift from Annakaisa Haapasalo, Finland. Domain deletion constructs of UBQLN1 were created using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).