American Society of Hematology

Figure 2

$Figure 2. Nuclear import and export of Liar. (A) Alignment of the NLS of Liar with the consensus NLS, critical arginine (R) and lysine (K) residues are highlighted in red. (B) Localization analysis of Liar NLS mutants. Wild-type Liar (Liar WT), NLS point mutants (Liar RR98AA, Liar KKLR111AALA), and the NLS-deleted Liar (LiarΔ98-114) were expressed as myc-tagged fusion proteins in COS-7 cells, and their subcellular localization was analyzed by confocal microscopy/immunofluorescence staining using anti-myc antibodies. The nucleus was identified by DNA counterstaining with Hoechst 33 258. (C) Alignment of the NES of Liar with the consensus NES; critical leucine (L) and phenylalanine (F) residues are highlighted in red. (D) Localization analysis of Liar the NES mutant. Wild-type Liar (Liar WT) and the NES point mutant (Liar LSL289ASA) were expressed and analyzed as described in panel B top. (E) Wild-type Liar-expressing cells were analyzed after treatment with Leptomycin B (+LB) for 2 hours (0.4 ng/mL). (F) Quantitation of nuclear Liar after treatment with Leptomycin B. (G) Subcellular fractionation analysis of Liar. Nuclear (N) and cytoplasmic (C) fractions of COS-7 cells expressing myc-tagged wild-type Liar (Liar WT), the NLS mutant (LiarΔ98-114), and the NES mutant (LiarLSL289ASA) of Liar were analyzed by Western blot analysis using anti-myc antibodies. Nuclear/cytoplasmic fractionation was confirmed by blotting for nucleolin (nuclear) and 14-4-4ζ (cytoplasm). (H) Nuclear and cytoplasmic distribution of Liar during the cell cycle. COS-7 cells expressing wild-type myc-tagged Liar were synchronized by thymidine block, then analyzed and quantitated as in panel B after release. Cell-cycle stages were determined by flow cytometry (propidium iodine staining) and morphology.$

Nuclear import and export of Liar. (A) Alignment of the NLS of Liar with the consensus NLS, critical arginine (R) and lysine (K) residues are highlighted in red. (B) Localization analysis of Liar NLS mutants. Wild-type Liar (Liar WT), NLS point mutants (Liar RR98AA, Liar KKLR111AALA), and the NLS-deleted Liar (LiarΔ98-114) were expressed as myc-tagged fusion proteins in COS-7 cells, and their subcellular localization was analyzed by confocal microscopy/immunofluorescence staining using anti-myc antibodies. The nucleus was identified by DNA counterstaining with Hoechst 33 258. (C) Alignment of the NES of Liar with the consensus NES; critical leucine (L) and phenylalanine (F) residues are highlighted in red. (D) Localization analysis of Liar the NES mutant. Wild-type Liar (Liar WT) and the NES point mutant (Liar LSL289ASA) were expressed and analyzed as described in panel B top. (E) Wild-type Liar-expressing cells were analyzed after treatment with Leptomycin B (+LB) for 2 hours (0.4 ng/mL). (F) Quantitation of nuclear Liar after treatment with Leptomycin B. (G) Subcellular fractionation analysis of Liar. Nuclear (N) and cytoplasmic (C) fractions of COS-7 cells expressing myc-tagged wild-type Liar (Liar WT), the NLS mutant (LiarΔ98-114), and the NES mutant (LiarLSL289ASA) of Liar were analyzed by Western blot analysis using anti-myc antibodies. Nuclear/cytoplasmic fractionation was confirmed by blotting for nucleolin (nuclear) and 14-4-4ζ (cytoplasm). (H) Nuclear and cytoplasmic distribution of Liar during the cell cycle. COS-7 cells expressing wild-type myc-tagged Liar were synchronized by thymidine block, then analyzed and quantitated as in panel B after release. Cell-cycle stages were determined by flow cytometry (propidium iodine staining) and morphology.

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