Analysis of expression of functional and non-functional human ORF2 protein in human cells. (A) Western blot analysis of proteins generated from expression plasmids containing codon-optimized, functional ORF2 endonuclease sequence (EN) and non-functional ORF2 endonuclease sequence transiently transfected in HeLa cells. Western blot analysis is performed with anti-human ORF2p monoclonal antibody (top), the previously described anti-human ORF2p endonuclease polyclonal antibodies (middle), and GAPDH antibodies (bottom). The non-functional ORF2 endonuclease sequence (EN 205, 230) has mutations resulting in expression of inactive ENp with D205A and H230A mutations. (B) The same experiment and analysis as in A, but using 293 cells. Western blot analysis of codon-optimized, non-functional ORF2 endonuclease sequences containing single inactivating mutations D205A or H230A (EN205 and EN 230, respectively) transiently transfected in 293 cells with anti-human ORF2 monoclonal antibody (top) or previously described anti-human ORF2p endonuclease polyclonal antibodies (middle). Expected EN protein size is 26 kDa. Control lane indicates cells transiently transfected with an empty vector; 25 and 37 kDa on the right indicate positions of molecular markers.