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. 2015 Jun 22;209(6):843–861. doi: 10.1083/jcb.201412050

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© 2015 Seybold et al.

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Figure 3. — Kar1 binds to the Sfi1 C-terminal region and Sfi1 binds to the Kar1 SPB duplication region. (A) Representation of Sfrs, tested for interaction with Kar1. (B) Kar1 binding to Sfrs from E. coli lysates. The fragments of A were expressed as N-terminal His tag fusions (top/input panel) and incubated with Sepharose-bound GST-Kar1ΔTMD (middle) or GST (bottom). Immunoblots with anti-His, anti-GST, and anti-Cdc31 are shown. (C) Kar1–Sfi1 interaction in S. cerevisiae cell lysates. A C-terminal construct of Sfi1 bearing the last five Cdc31 binding repeats (CT+5) was expressed alone or together with TAP-Kar1ΔTMD or TAP tag. After IgG Sepharose incubation, proteins were analyzed by immunoblotting with anti-TAP (top) or anti-Sfi1 (bottom) antibodies. (D) Cartoon of Kar1 fragments used in E. Hatched box, TMD domain. (E) Sfi1 binding to Kar1 fragments. GST-tagged Kar1 fragments from E. coli were incubated with Sfi1 CT+5 as in B. Immunoblots with indicated antibodies are shown. aa, amino acid; NT, N terminus; CT, C terminus; Sfr, Sfr.