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. 1999 Jun 14;145(6):1145–1152. doi: 10.1083/jcb.145.6.1145

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RS domain–mediated nuclear import in vitro. Digitonin-permeabilized HeLa cells were incubated with the import substrate GST-ASF/SF2 RS along with either buffer (−Cytosol) or rabbit reticulocyte lysate (all other panels). An ATP-regenerating system was included in all assays except the −ATP experiment. In addition to omitting the ATP-regenerating system, the −ATP import assay included apyrase at 2.5 U/μl. Where indicated, RanQ69L (2 μM), WGA (0.2 mg/ml), or an importin β fragment containing amino acids 45–462 (Impβ ΔN44, 1 μM) was added to the import mixture. Import of GST-ASF/SF2 RS was detected by immunofluorescence microscopy using an anti-GST antibody.