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. 2020 Aug 31;19(11):1896–1909. doi: 10.1074/mcp.RA120.002050

Fig. 3.

Fig. 3.

Erg25 is a proteasome substrate. A, Cell lysates were prepared from Pre8:GFP Δpdr5 or Δpdr5 yeast strains expressing C-terminally HA epitope tagged Erg25 (Erg25-HA) or harboring a vector control. Immunoprecipitations (IP) in the presence of MG132 were performed with anti-HA affinity matrix as described under Experimental Procedures. Immunoblots (IB) after precipitation of cell lysates with anti-HA or anti-GFP are indicated. *nonspecific band. B, Cell lysates from Δpdr5 yeast expressing Erg25-HA and myc-ubiquitin grown in the presence or absence of 50 μm MG132 were subjected to lysis under denaturing conditions (to preclude the isolation of nonspecific partners) and immunoprecipitated with anti-HA affinity matrix. Precipitated proteins were immunoblotted with anti-HA (Erg25) and anti-ubiquitin. Data represent the means of 5 experiments, ± S.D. *, p < 0.05. C, Cycloheximide chase assays were performed as described under Experimental Procedures in untransformed Δpdr5 yeast strains incubated in the absence or presence of MG132 and immunoblots were probed with anti-Erg25 antisera. Data represent the means of 4-6 experiments, +/-S.D. *, p ≤ 0.05.