Figure 4

Analysis of expression of functional and non-functional full-length and truncated human ORF2 proteins in human cells. (A) Schematic of L1 ORF2 protein. The following ORF2p domains are listed: Endonuclease domain (EN), Z domain (Z), Reverse Transcriptase domain (RT), and the Cysteine-Rich domain (Cys). Amino acid boundaries of each domain are listed. (B) Western blot analysis of the full-length and truncated ORF2 proteins generated from expression plasmids containing codon-optimized, functional full-length ORF2 (ORF2) and C-terminally truncated ORF2 sequences transiently transfected in 293 cells with monoclonal antibody. Control lane indicates cells transiently transfected with an empty vector. (C) Western blot analysis of proteins with two inactivating mutations are labeled as 205, 230, single mutants are labeled as 205 or 230 generated from expression plasmids containing codon-optimized, mutant of the constructs described in A transiently transfected in 293 cells with monoclonal antibody. Constructs containing two inactivating mutations are labeled as 205, 230, single mutants are labeled as 205 or 230. (D) Western blot analysis of the same samples described in A with polyclonal anti-ORF2p antibodies. (E) Western blot analysis of the same samples described in B with anti-human ORF2p polyclonal antibodies. GAPDH is used as a loading control; 50 to 150 kDa and 37 kDa on the right indicate molecular markers. Arrows denote bands of expected molecular weights for each construct listed.