Figure 2.
Hmg2p was ubiquitinated. (A) (Left) Strains expressing myc-Ub along with high levels (HIGH) or low levels (low) of Hmg2p were assayed for ubiquitination by IP of Hmg2p followed by immunoblotting for coprecipitated myc-tagged-Ub (myc-Ub). (Right) myc-Hmg2p was the result of subjecting an isogenic strain that expressed myc-Hmg2p but not myc-Ub to the identical assay. Arrows represent (single or double- sided) the mobility of Hmg2p (Mr 115). Line segments represent the mobility of a 190-kDa protein standard. (B) Comparison of myc-Ub coprecipitation in isogenic strains expressing comparable levels of degraded Hmg2p (lane 2) or stable Hmg1p (lane 1). IP lanes are anti-myc immunoblots of the immunoprecipitated proteins. The lanes are heavily exposed to indicate the extreme difference obtained with the distinct isozymes. Lysate indicated cell lysates that were immunoblotted for myc immunoreactivity to assess myc-Ub induction. (C) Effect of ubc7::LEU2 on Hmg2p ubiquitination. Wild-type (RHY506) and ubc7::LEU2 (RHY511) strains were transformed with YEp112, a plasmid analogous to YEp105 that expresses HA-Ub. Lysates were immunoprecipitated then immunoblotted either with monoclonal 12CA5 to detect HA-Ub (Left, 4/5 total) or 9E10 to detect myc-Hmg2p (Right, 1/5 total). The lysates displayed identical induction of the HA-tagged Ub (not shown)