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. 1997 Apr 29;94(9):4463–4468. doi: 10.1073/pnas.94.9.4463

Figure 3.

Figure 3

Bem1 binds preferentially to GDP-bound Bud1 in vitro. Purified GST–Bud1, GST–Bud1T35A, and GST (approximately 750 nM) were preincubated with 1 mM GDP or GTP[γS] and then incubated with six-histidine-tagged Bem1 (15 nM). Bem1 was incubated with GTP[γS]–Bud1 (lane 1), GDP–Bud1 (lane 2), GTP[γS]–Bud1T35A (lane 3), GDP–Bud1T35A (lane 4), GST preincubated with GTP[γS] (lane 5), or GDP (lane 6) or with GST–Bud1 (lane 7) or GST–Bud1T35A (lane 8) that had not been preloaded with any nucleotides. Proteins bound to glutathione-Sepharose beads were collected and analyzed by SDS/PAGE. Association of Bem1 was determined by immunoblotting with polyclonal antibodies against Bem1 (ref. 30 and Kathy Corrado and J.R.P., unpublished results). A negative control is shown in lane 9, in which no GST–Bud1 or GST was added.