Exo70p and Bem1p interact with each other in vitro. (A) GST-Exo70p or GST-Sec22NTp were immobilized on glutathione beads and mixed with purified C-terminal His₆-tagged Bem1p, Vam7p, or N-terminal His₆-MBP–tagged Sec9CTp. Bound proteins were detected with anti-His antibody. (B) GST, GST-Bem1p, or GST-Gea2ΔNp were pre-immobilized on glutathione beads and mixed with purified His₆-Exo70p. Bound GST fusion proteins were detected with CBB staining and associated His₆-Exo70p was detected with anti-His antibody. (C) GST-Exo70p was immobilized on glutathione beads in concentrations ranging from 0 to 4.59 µM. It was then incubated with 25 nM Bem1p-His₆. After pelleting the beads, the amount of Bem1p-His₆ remaining in the supernatant was determined by Western blotting using anti-His antibody and quantified with an Odyssey imaging system. The K_d was calculated with Origin software. The data shown are from a single representative experiment out of three independent repeats.