Figure 3.

Add66p is cytosolic. (A) add66Δ strains were transformed with a control plasmid or with a plasmid engineered for the constitutive expression of Add66p-myc. Cell lysates (L) were subjected to 16,000 × g and 150,000 × g centrifugations. Total proteins in the pellets (P1 and P2) and supernatants (S1 and S2) were resolved by SDS-PAGE, and then they were analyzed by Western blot analysis with anti-myc, anti-Sec61p (ER membrane protein), anti-Sse1p (a primarily cytosolic protein; Goeckeler et al., 2002), and anti-Cim5p (a regulatory subunit of the 26S proteasome with cytosolic and ER membrane subcellular localizations) antisera. (B) Indirect immunofluorescence of add66Δ strains transformed with the plasmids described in A were stained with 4,6-diamidino-2-phenylindole (nuclear staining), and then they were probed with anti-BiP (ER perinuclear and peripheral staining) and with anti-myc antisera, and signals were detected as described in Materials and Methods.