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Fig. 4 | Mobile DNA

Fig. 4

From: Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein

Fig. 4

Mutation of charged residues in the RRM and CTD of the full-length ORF1 results in a shift of the truncated ORF1p into the cytoplasmic fraction. a Schematic of part of the RNA recognition motif (RRM) and C-terminal domain (CTD) of ORF1. Positions of domains shown are approximate. Human ORF1p (top) is aligned to mouse ORF1p (bottom) using Lipman-Pearson method. Black arrows denote the amino acid position of the lysine residues that were mutated to alanine residues in the human ORF1. The lysine mutant contains all 14 lysine residues (all black arrows) mutated to alanine residues. b Western blot analysis of full-length human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with human-specific ORF1 polyclonal antibodies (hORF). Calregulin (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. c Schematic for (d) and (e). Green arrows denote the amino acid position of the lysine residues that were mutated to alanine residues in the human ORF1. Human ORF1p (top) is aligned to mouse ORF1p (bottom) using Lipman-Pearson method. Black arrows denote the amino acid position of the lysine residues that were mutated to alanine residues in the human ORF1. “(5KA)” denotes a construct in which the five residues listed (227,229, 237, 243 and 245) were mutated from lysine to alanine residues. d Western blot analysis of full-length human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with human-specific ORF1 polyclonal antibodies (hORF1). Calregulin (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. “(5KA)” denotes a construct in which the five residues (227,229, 237, 243 and 245) were mutated from lysine to alanine residues (227,229, 237, 243 and 245). e Western blot analysis of full-length human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with human-specific ORF1 polyclonal antibodies (hORF1). Calregulin (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. “(5KA)” denotes a construct in which the five residues (227,229, 237, 243 and 245) were mutated from lysine to alanine residues (227,229, 237, 243 and 245) in addition, other residues that were also mutated in the construct are listed above each lane

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