The nucleoporin Nup159 is a novel Bfa1 interactor.

(A–D) Stationary phase cultures in YPAD were diluted to OD600 = 0.2 in fresh medium and then grown for 6 h at 26°C. (A, B) Co-immunoprecipitation analysis in cells simultaneously expressing Nup159-GFP and either 3HA-Bfa1 or Bub2-3HA in different genetic backgrounds. Cells exclusively expressing 3HA-Bfa1, Bub2-3HA, or Nup159-GFP were included as controls. Western blot gel images for 3HA-Bfa1, Bub2-3HA, and Nup159-GFP are shown for both the input (INPUT) and the immunoprecipitated (Co-IP) samples. The Co-IP efficiency for 3HA-Bfa1 relative to the corresponding control with untagged Nup159 (-) and referred to the strain used as a reference (×1.00) in (A) is indicated in each case. Each sample in (A) was separated from the rest with an empty well to discard any residual transfer between lanes. Experiments were carried out thrice (n = 3) and a representative image is shown. (C, D) BiFC analysis of Bfa1-VC interaction with VN-tagged nucleoporins. (C) Illustrative image displaying a positive BiFC interaction (Bfa1-VC/Nup159-VN, in green and marked with an arrow) and SPB localization (Spc42-mCherry, in red). Nuclear morphology (DAPI, in blue), PhC, and merged images are also shown. (D) Quantification of the percentage of cells displaying positive BiFC interaction. Data are the average of 3 samples (n = 3; 100 cells/each) and are available in S1 Data. Error bars represent SD. BiFC, bimolecular fluorescence complementation; GFP, green fluorescent protein; PhC, phase-contrast; SPB, spindle pole body.