Fig. 2

Vimentin is an interacting partner for Nup88. A GFP-coprecipitation assay was performed using cell lysates containing either GFP or GFP-tagged Nup88. Coprecipitates recovered were analyzed by SDS-PAGE and CBB protein staining (a). Specific interaction of vimentin with GFP-tagged Nup88 was confirmed by immunoblotting using an antibody against pan-vimentin. Inputs are 1.5% for vimentin and 1.0% for GFP constructs (b). GFP-coprecipitation assay for the vimentin interaction with Nup88 (c), Nup214 (d), Nup358 (e) and Nup50 (f). Inputs are 8.3% for both Nup88-FLAG and GFP constructs (c), 5.0% for Halo-tagged Nup214 (Halo-Nup214) and 3.3% for GFP constructs (d), 4.0% for Halo-tagged Nup358 (Halo-Nup358) and 3.0% for GFP constructs (e), and 3.7% for both GFP constructs and vimentin (f), respectively. Arrows indicate GFP-tagged vimentin (c–e) and GFP-tagged Nup50 (f), respectively. Schematic of GFP-tagged Nup88 constructs used in Fig. 2h. Amino acid residues of Nup88 are numbered (g). Determination of Nup88’s region responsible for vimentin binding. Cell lysates containing the GFP-tagged proteins described in Fig. 2g were analyzed. Inputs are 1.0% for GFP constructs and 1.3% for vimentin, respectively (h). GST-pulldown assay to examine the direct binding of recombinant GST-tagged Nup88 to recombinant vimentin. Inputs are 8.0% for both vimentin and GST constructs, respectively (i)