A re-evaluation of LINE-1 ORF2 expression in LNCaP prostate cancer cells.

We previously examined expression of Long Interspersed Element-1 (LINE-1) in a variety of prostate cancer cells including hormone-dependent LNCaP cells. These studies demonstrated expression and sub-cellular localization of LINE-1 proteins, ORF1p, with antibody 4H1, and ORF2p, with antibody chA1-L1. Here we conduct immunoprecipitation/mass spectrometry analysis using chA1-L1 antibody against ORF2p in LNCaP cells. Our results indicate that antigens detected by the antibody include the transcriptional regulator BCLAF1. We show that chA1-L1 recognizes BCLAF1 using siRNA knockdown and overexpression of a tagged BCLAF1. We also show that chA1-L1 antibody recognizes ORF2p in HEK293 cells overexpressing LINE-1. Further, analysis of ORF2p (chA1-L1) and BCLAF1 foci using immunofluorescence in LNCaP cells showed significant colocalization. Overall, our findings indicate that chA1-L1 antibody recognizes both BCLAF1 and ORF2p but the majority of antigen recognized in LNCaP cells is BCLAF1.