Fig. 1

Generation and characterization of α-human L1 ORF1p (RRM) antibody: a Diagram of full-length active human L1 retrotransposon. L1 encodes two proteins (ORF1p and ORF2p). ORF1p is characterized by three distinct domains: coiled-coil (CC), RNA Recognition Motif (RRM) and Carboxy Terminal Domain (CTD). The RRM domain alone (amino acids 157–252) was sub-cloned in pET bacterial expression vector. b SDS-PAGE of E.coli expressed pET human L1 ORF1p (RRM) peptide. Lane 1: soluble fraction, Lane 2: elution, Lane 3: flow through. A contaminate protein (less than 1% of the total amount) with molecular mass of around 70 kDA was also eluted with RRM domain. The eluted ORF1p (RRM) peptide was injected into a rabbit for generating α-hORF1p (RRM) antibody. c Immunoblot analysis of cell lysates from diverse murine and human cell lines using the α-hORF1p (RRM) antibody.Lane1: NIH3T3 (mouse embryonic fibroblast), Lane 2: DU145 (human prostate cancer cell line), Lane 3: HeLa (human epithelial cancer), Lane 4: MCF-7 (human breast cancer) and Lane 5: HEK293T (human embryonic kidney). Panel 2- immunoblot with α-GAPDH (loading control). d Immunoblot analysis of L1 ORF1p expression in pcDNA-ORF1F-transfected HeLa cells. Using the α-hORF1p antibody, ORF1p expression is detected in transfected HeLa cells but not detectable in untransfected HeLa cells. (Construct: ORF1F; ORF1 tagged with C-terminal FLAG cloned in pcDNA vector); Panel 2- immunoblot with α-GAPDH (loading control). e Immunoblot analysis of ORF1p expression in pcDNA-ORF1F-transfected HEK 293 T cells. Lane 1: transfected, Lane 2: Untransfected. Panel 1: immunoblot with α-human L1 ORF1p (RRM), Panel 2: with α-FLAG and Panel 3: with α-GAPDH (loading control) f Quantification of ORF1p amount by image J software (https://imagej.net/ImageJ) in total lysate in from MCF-7 and HEK 293 T cells