FIGURE 6:

The 611–950 segment of Hsl1 binds directly to assembled septin filaments in vitro. (A) Diagram of the primary structure of the His₆-MBP-Hsl1(611–950) fusion protein expressed in and purified from E. coli as described in Materials and Methods. (B) Cosedimentation assay. Cdc11-capped hetero-octamers, purified as described in Materials and Methods, alone (lanes 1–4), purified His₆-MBP-Hsl1(611–950) alone (lanes 5–8), a 1:2 mixture of Cdc11-capped hetero-octamers and His₆-MBP-Hsl1(611–950) (lanes 9–12), purified His₆-MBP alone (lanes 13–16), or a 1:2 mixture of Cdc11-capped hetero-octamers and His₆-MBP (lanes 17–20) were incubated in either high-salt buffer (H) or low-salt buffer (L) and then subjected to high-speed centrifugation and separated into supernatant (s) and pellet (p) fractions, the contents of which were then resolved by SDS–PAGE and analyzed by staining with InstantBlue dye. (C) Samples of the indicated mixtures from B were applied to carbon-coated copper grids, stained, and examined by EM, and representative images were recorded. Scale bar, 100 nm.