Constructs

pFLAG-Derlin-1 and pFLAG-Derlin-2 were constructed by inserting the cDNA fragments encoding the open reading frames (ORF) of human Derlin-1 and Derlin-2, respectively, amplified by PCR and cut with HindIII/XhoI, to the HindIII/SalI sites of pFLAG-CMV-6a. pFLAG-RNF5 was constructed by inserting the cDNA fragments encoding the ORF of human RNF5 to the BglII/SalI sites of pFLAG-CMV-6C. In the pGEX-RNF5N plasmid, the cDNA fragment encoding the N-terminal 117 aa of human RNF5 was amplified by PCR, cut with BglII/XhoI, and then inserted into the BamHI/SalI sites of pGEX-4T-3. In the pGEX-Hrd1C plasmid, the BclI/NotI fragment of human Hrd1 cDNA, encoding the C-terminal portion (aa235–617) of human Hrd1 was inserted into the BamHI/NotI sites of pGEX-5X-1. pGEX-praja1 was previously published (Lorick et al., 1999).