Analysis of subcellular localization of hORF1p expressed from vectors containing wild type sequences.

<p><b>A</b>. (top) Western blot analysis of subcellular localization of ORF1p (blue rectangle labeled as hORF1) expressed from human full-length L1 (hL1wt, 40 kDa), human ORF1 wt (hORF1wt, 41 kDa) or hORF1co (40 kDa) vectors transiently expressed in HeLa cells. Human ORF1p is detected in nuclear (N) and cytoplasmic (C) fractions with human-specific ORF1p polyclonal antibodies. GAPDH (cytoplasmic marker) and Lamin A (nuclear marker) are used as loading and cell fractionation controls. 37 kDa is a molecular marker. Control lanes indicate cells transfected with empty vector. (bottom) Quantitation of western blot results: each construct expressing hORF1p was normalized to its respective GAPDH and Lamin A loading controls. The relative amounts of nuclear and cytoplasmic ORF1p were calculated as a fraction of the relative total ORF1p detected. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>B</b>. The same experiment and analysis as in A was performed in NIH-3T3 cells. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>C</b>. Subcellular localization of mORF1p (green rectangle labeled as mORF1) transiently expressed from the full-length mouse L1 (mL1wt) or codon-optimized mORF1 (mORF1co) in HeLa or NIH-3T3 cells using mouse specific polyclonal antibodies. 50 kDa is a molecular marker.