(A) Cells co-expressed a galactose-inducible GST-cyclin (or vector) with V5-tagged Ste5. After galactose induction, GST fusions and co-bound proteins were captured with glutathione-Sepharose. Bound and input proteins were analyzed by immunoblots.
(B) Cells expressed V5-tagged Cln2 (wt or lpd) and galactose-inducible GST fusions to full-length proteins or N-terminal fragments. Ste5* is a hybrid fragment [22, 29], in which the Cln2-docking site in the Ste20 N-terminus is replaced with one from Ste5. Grr1ΔF lacks its F-box, to prevent it from driving degradation of Cln2 [25]. Also see Figure S3.