Figure 10.

The amino-terminal region of Nup116p is required for in vitro interaction with Gle2p. Gle2p-HA was cotranslated in vitro with the indicated Nup116p and Nup145p polypeptides (see Figure 1A). (A) Autoradiograph of a 7% SDS-polyacrylamide gel with aliquots of the respective cotranslation input fractions used for immunoprecipitation in B. Lane 1 is a translation of Gle2p alone. The position of the Gle2p doublet is noted by the arrow. The upper bands correspond to the respective Nup116p or Nup145p products. (B and C) The ³⁵S-labeled polypeptide mixtures were immunoprecipitated with affinity-purified rabbit polyclonal anti-GLFG antibody (B) or mAb12CA5 (anti-HA/Gle2p, C) and protein A– Sepharose beads. The bound fraction was eluted by boiling in SDS sample buffer and separated by electrophoresis on either a 7% (B) or 13% (C) SDS-polyacrylamide gel. ³⁵S-labeled proteins were detected by autoradiography. Molecular mass markers are noted in kDa.