Fig. 4. Sem1 contributes to RP stability. (A) Immunoprecipitation analysis of proteasomes. Flag-His₆-Pre1 was expressed in YPH499 or sem1 cells. Extracts were prepared after cells were incubated at 37°C for 6 hours, and were immunoblotted with the indicated antibodies (lanes 1-3). In lanes 4-6, anti-Flag immunoprecipitation was followed by immunoblotting. Inputs (lanes 1-3) were 1/3 to 1/2 of the amounts used in lanes 4-6. (B) Proteasome stability in sem1 cells. Cell extracts at 30°C were resolved by nondenaturing PAGE either directly (lanes 1, 2) or following affinity purification of Flag-Pre1-containing proteasomes (lanes 3-6). CP-containing species were detected by an in-gel peptidase assay using the Suc-LLVY-AMC fluorogenic substrate and UV illumination of the gel. Extracts from cells grown at 37°C showed a similar pattern. (C) Glycerol gradient analysis of sem1 proteasomes. Glycerol gradient fractions were evaluated for the proteasomes by immunoblot analysis with antibodies against the indicated proteins (top) and by CP peptidase activity assays (bottom). The anti-CP antibody was MCP231, which recognizes multiple subunits. Asterisk indicates cross-reacting protein.