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. 1999 Mar 22;144(6):1097–1112. doi: 10.1083/jcb.144.6.1097

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The Nup98-Nup96 precursor is proteolytically processed in vitro and in vivo. (A) Schematic representation of the Nup98-Nup96 precursor protein constructs used in the in vitro and in vivo expression assays. Each protein contains a myc-epitope tag (shown in gray) at its NH₂ terminus. (B) In vitro transcription/translation reactions. Protein constructs were transcribed and translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. Reactions were separated by SDS-PAGE and analyzed by autoradiography. Reactions were performed with no DNA (lane 1), wild-type Nup98-Nup96 precursor (lane 2), mutant Nup98-Nup96 precursor (F863S/Y866R) (lane 3), Nup98 (1–863; lane 4), and Nup96 (lane 5). Molecular mass markers are indicated on the left. (C) In vivo expression of the wild-type and mutant Nup98-Nup96 precursors. HeLa cells were transfected with no DNA (lane 1), wild-type Nup98-Nup96 precursor (lane 2) and mutant Nup98-Nup96 precursor (F863S/Y866R) (lane 3). 24 h after transfection, cells were lysed in sample buffer and analyzed by immunoblot analysis using an anti-myc epitope antibody.

The Nup98-Nup96 precursor is proteolytically processed in vitro and in vivo. (A) Schematic representation of the Nup98-Nup96 precursor protein constructs used in the in vitro and in vivo expression assays. Each protein contains a myc-epitope tag (shown in gray) at its NH₂ terminus. (B) In vitro transcription/translation reactions. Protein constructs were transcribed and translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. Reactions were separated by SDS-PAGE and analyzed by autoradiography. Reactions were performed with no DNA (lane 1), wild-type Nup98-Nup96 precursor (lane 2), mutant Nup98-Nup96 precursor (F863S/Y866R) (lane 3), Nup98 (1–863; lane 4), and Nup96 (lane 5). Molecular mass markers are indicated on the left. (C) In vivo expression of the wild-type and mutant Nup98-Nup96 precursors. HeLa cells were transfected with no DNA (lane 1), wild-type Nup98-Nup96 precursor (lane 2) and mutant Nup98-Nup96 precursor (F863S/Y866R) (lane 3). 24 h after transfection, cells were lysed in sample buffer and analyzed by immunoblot analysis using an anti-myc epitope antibody.