Figure 10.

Ubiquitination of wild-type and mutant forms of Ste6p. Strains expressing STE6::HA and myc-tagged ubiquitin were used to examine the extent of ubiquitination of various forms of Ste6p. Protein extracts prepared from these strains were subjected to immunoprecipitation with the 12CA5 (anti-HA) antibody to immunoprecipitate Ste6p. The immunoprecipitates were separated on a 8% SDS gel and transferred to nitrocellulose. (A) The membrane was probed with the 9E10 (anti-myc) antibody to detect ubiquitinated forms of Ste6p. (B) The membrane was stripped and reprobed with the anti-HA antibody to detect total amounts of Ste6p present in the extracts. The bar at left indicates identical positions on panels A and B. Strains are SM3548 ([2μ STE6::HAc, 2μ ubi::myc]), SM3668 ([2μ ste6–13::HAc, 2μ ubi::myc]), SM3550 ([2 μ ste6–166::HAe, 2μ ubi::myc]), and SM3669 ([2μ STE6 (untagged), 2μ ubi::myc]).