Fig. 3. Sem1 is a component of the RP lid. (A) Sem1 is tightly associated with the RP. Endogenous PRE1 (CP) or RPT1 (RP) genes were replaced with alleles that expressed the corresponding proteins with Flag-His₆ tags. Extracts from yeast expressing Flag-His₆-Pre1 or Flag-His₆-Rpt1 were precipitated with anti-Flag antibody, followed by immunoblotting with antibodies to the indicated antigens. Inputs (lanes 1-6) represent 15% (for -Sem1) or 45% (for -Rpt1, -20S and -Flag) of the amounts used for precipitation (lanes 7-12). (B) Co-elution of Sem1 in proteasome-containing gel-filtration-column fractions. Anti-Sem1 (top), anti-Rpt6 (middle), and anti-CP subunit (MCP231) plus anti-HA antibodies (bottom) were used for immunoblotting. Size standards are shown at the bottom. (C) Sem1 is tightly associated with the lid. The lid and base were salt eluted from RPs immobilized with protein-A tags on either the base (lanes 1-4) or the lid (lanes 5-8), followed by immunoblotting with antibodies as indicated. Proteins run on the gel were bound to the IgG resin prior to batch salt washes (lanes 1, 5); eluted with 0.3 M NaCl (lanes 2, 6); eluted with 1 M NaCl (lanes 3, 7); or remained after salt washes (lanes 4, 8). Positions of molecular weight standards are indicated on the left.