Pcmv6 entry vector

Full-length cDNA encoding human AIG1 (GE Healthcare, in pOTB7 vector) was cloned into the pCMV6-Entry vector with C-terminal Myc and DDK tags. (Sense primer: 5'-GGGCGGCCGGGAATTCGCGAACATGG-3'; Antisense primer: 3'-AAGCCTAAATTGGAAACGCGGCCGCTTTA-5'). Full-length cDNA encoding for human ADTRP in the pCMV6-XL5 vector was purchased from Origene. Full-length cDNA constructs encoding for mouse AIG1 in the pCMV-Sport6 vector, rat AIG1 in pExpress-1 vector, and mouse ADTRP in the pCMV-Sport6 vector were purchased from GE Life Sciences. To recombinantly express AIG1 or ADTRP, HEK293T cells were grown to 40% confluence in a 10 cm tissue culture plate and transiently transfected with 4 μg of the desired construct using polyethyleneimine ‘MAX’ (MW 40,000, PEI; Polysciences, Inc.) as the transfection reagent per the manufacturer’s protocol. ‘Mock’ transfected cells were transfected with 4 μg of empty vector. 48 h after transfection, cells were washed with PBS (3x), harvested by scraping, and lysed by sonication in PBS. The membrane and soluble fractions were separated by ultra-centrifugation at 100,000 g for 45 min at 4 °C. Protein concentrations were measured using the DC Protein Assay kit (Bio-Rad). Aliquots were flash-frozen and stored at −80 °C for further use.