Fig. 3.

Characterization of the RLLL degron. a The solubility of the reporter proteins was analyzed by fractionating cell extracts into soluble supernatant (S) and insoluble pellet (P) fractions by centrifugation. The protein levels were analyzed by SDS-PAGE and Western blotting using antibodies to the HA-tag in the reporter. Pgk1 served as a loading control for the soluble protein fractions, while Pma1 served as loading control for the insoluble protein fractions. b Myc-tagged Ssa1 (Hsp70) was co-immunoprecipitated (co-IP) with the indicated GFP fusions using GFP-trap resin from cultures treated with bortezomib. ATP was not added to the buffers used for the co-IPs. The precipitated protein was analyzed by SDS-PAGE and blotting with antibodies to myc and GFP. c The protein levels of the RLLL degron were compared in the indicated yeast strains by SDS-PAGE and blotting for the HA-tag on the reporter. A Ponceau S staining of the membrane is included as loading control. d The dependence of E3 ligases for targeting the RLLL degron was analyzed by growth assays on solid media using the indicated null mutants. Wild-type cells transformed with the reporter vector alone were included for comparison