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. 2014 Oct 28;5:5339. doi: 10.1038/ncomms6339

Figure 2. Kinesin-5 Cut7 binds the γ-TuRC MTOC.

Figure 2

(a) Kinesin-5 and kinesin-14 constructs used in Fast Protein Liquid Chromatography. V5-tagged Cut7 and two truncation constructs were used, in addition to one FLAG-Pkl1 truncated construct that retains full Pkl1 activity. Cut7 constructs are V5-tagged full-length Cut7 (aa 1–1,085), Cut7-Head-Stalk (Cut7HS, aa 1–888) and Cut7-Stalk-Tail (Cut7-ST, aa 443–1,085). (b) Western blot profiles of whole-cell extracts fractionated by Separose 6 using FPLC. (c) Western blots of Cut7 constructs immunoprecipitated from whole-cell extracts using anti-V5 magnetic beads with empty strain negative controls. (d) Cartoon diagram of 6-His tagged Pkl1 Tail peptide co-immunoprecipitation assay using magnetic beads with His affinity and FPLC fraction 15. (e) Pkl1 Tail peptide co-immunoprecipitation of γ-TuRC core subunits and V5-Cut7ST using a short Pkl1 Tail peptide (PγT). Mutated peptide PγM has significantly reduced interaction with the fission yeast γ-TuRC. The anti-HA antibody detects the HA-tagged γ-TuRC protein Alp4.