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. 2001 Mar;12(3):521–538. doi: 10.1091/mbc.12.3.521

Figure 3.

Figure 3

Sed5p selectively forms ternary and quaternary complexes in vitro with the SNAREs Sft1p, Bet1p, Gos1p, Bos1p, Ykt6p, and Sec22p. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and recombinant (His)6-tagged SNAREs detected by immunostaining with the anti-(His)6 antibody BMG-His-1. Ternary complexes: GST-Sft1p was mixed with various combinations of two different (His)6-tagged SNARE proteins as indicated (A). GST-Bet1p was mixed with various combinations of two different (His)6-tagged SNARE proteins as indicated (B). Quaternary complexes: Either GST-Bet1p or GST-Sft1p was mixed with (His)6-Sed5p and various combinations of two other (His)6-tagged SNARE proteins as indicated (C). Immunoblot analysis of aliquots of soluble lysates from bacteria expressing the (His)6-SNAREs used in this experiment (corresponding to ∼5% of input) (D). (E) Longer exposure of a subsection of B reveals some association of Ykt6p with [Bet1p + Sed5p] and of Bos1p with [Bet1p + Sed5p]. Note that in the absence of Sed5p no detectable binding is observed between either GST-Sft1p or GST-Bet1p and other SNAREs (A and B, lanes 5–8).