Journal: Molecular and Cellular Biology
Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export
Figure Lengend Snippet: Interaction of nuclear transport receptors with Nup50. (A) NRK cells were solubilized in NP-40 buffer, and extracts were immunoprecipitated with anti-Nup50 antibodies (antibody 1). Material bound to the antibody beads (b) and the unbound material (ub) were electrophoresed on an SDS gel and analyzed by immunoblotting with anti-CRM1, anti-CAS, anti-importin β, or anti-lamin antibodies. Note that the sample loaded in the bound lanes represents sevenfold more cell equivalents than the material in the unbound lanes. (B) Sepharose beads coupled with a Nup50 fragment (Nup50) or with BSA were incubated with recombinant CRM1, CAS, or importin β in the absence or presence of RanGMP-PNP or cytochrome c coupled with NES peptides (cc-NES) as indicated. Samples were analyzed by immunoblotting with antibodies recognizing the His 6 tag of the recombinant receptors. An equivalent amount of the bound material was loaded in all cases. Moreover, the input material contained nearly equal quantities of all transport receptors, as determined by protein analysis. (C) NRK cells were incubated at 37°C for 4 h in growth medium containing 100 nM leptomycin B (+LMB) or lacking leptomycin B (−LMB). They were then fixed with methanol-acetone (see Materials and Methods) and examined by confocal microscopy after double immunofluorescence staining with anti-Nup50 (antibody 1) or RL1, as indicated.
Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).
Techniques: Immunoprecipitation, SDS-Gel, Incubation, Recombinant, Confocal Microscopy, Double Immunofluorescence Staining