Fig. 2
From: LINE-1 ORF2p expression is nearly imperceptible in human cancers
Production of monoclonal ORF2p antibodies. a Expression constructs used to generate antigens for ORF2p antibody production. b Coomassie-stained protein electrophoresis gels illustrating purity of ORF2p antigens used in antibody generation. c Immunization strategy to produce rabbit monoclonal antibodies. d Western blot detection of overexpressed ORF2p-3xFlag obtained from HEK-293TLD cells transfected with pLD561 (shown in panel a) using 5 different monoclonal antibodies (Ab) compared to anti-Flag. e Immunoprecipitation of ORF2p-3xFlag using 3 antibodies. f Immunofluorescence imaging of HEK-293TLD cells expressing ORF2p-3xFlag showing co-localization with anti-Flag antibody. g Immunohistochemistry of HEK-293TLD cells expressing ORF2p-3xFlag with 4 monoclonal antibodies compared to anti-Flag. h Above, overview of PhIP-Seq. A phage library expresses protein epitopes from the protein-coding genome, which are affinity purified with ORF2p antibodies. DNA sequences are then isolated and sequenced to identify the genes encoding the peptides. Below, results from five monoclonal antibodies targeting ORF2p. In each instance, the greatest affinity of the ORF2p monoclonal antibodies is for peptides encoded by L1Hs ORF2p peptides. EN = endonuclease, RT = reverse transcriptase, MBP = mannose binding protein, SUMO = small ubiquitin-like modification