Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Ankle1 shuttles between nucleus and cytoplasm. a Schematic representation of Ankle1’s domain organization depicting predicted ankyrin repeats, the LEM domain and a GIY-YIG nuclease domain. Putative nuclear export sequences (NES1, NES2) and nuclear localization sequences (NLS1, NLS2), identified in silico are indicated. b Immuno-fluorescence analysis of ectopic Ankle1-V5 in U2OS cells without or following a 3 h treatment with 50 nM leptomycin B, an inhibitor of CRM1-mediated export. Cells were stained with antibodies to V5, and DNA with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: In Silico, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Localization of Ankle1 fragments containing different domains and export and import signals. Localization of GFP-tagged Ankle1 truncation constructs ectopically expressed in U2OS ( a ) and HeLa ( b ) cells was determined by confocal fluorescence microscopy. Molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. Cells were fixed after 3 h of mock or leptomycin B treatment. DNA was counterstained with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Construct, Fluorescence, Microscopy

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Mutation analyses identify NES2 and NLS2 as the predominant sequences controlling nucleo-cytoplasmic shuttling of Ankle1. a , b , d , e U2OS cells were transiently transfected with Ankle1-V5 carrying point mutations in NLS or NES sequences and either mock-treated or treated with leptomycin B for 3 h and processed for confocal immunofluorescence analyses using antibodies to V5 and DAPI to detect DNA. Scale bars: 10 μm. c Mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type Ankle1-V5, Ankle1-NES1mut-V5 or Ankle1-NES2mut-V5 were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. Data were obtained from three independent experiments and analyzed using Student’s t -test. Ankle1-NES1, P = 0.002; Ankle1-NES2, P = 5.4E-21; n = 50; 15–17 cells each from three independent experiments

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Expressing