Figure 3.

Detection of SDS-insoluble aggregates in the supernatant fraction of lysates of the GFP^N104-expressing cells by the filter trap assay. Cells were grown to log phase in raffinose-containing media, induced for GFP^C and GFP^N104 expression by addition of galactose, and harvested for lysis after 5 hours. (A) Cell lysates were supplemented with SDS to a final concentration of 2% and boiled for 10 min at 100°C. Cell lysates were diluted with buffer H supplemented with 2% SDS according to the number of cells of each sample before being loaded onto a nitrocellulose membrane and subjected to Western blotting by using the monoclonal anti-Myc antibody 9E10. The number of cells loaded in the first well is indicated, with two-fold dilution in the subsequent wells. (B) Supernatant fractions were prepared from cell lysates by centrifugation at 16,100 xg for 2 min. Supernatant fractions were diluted and loaded onto cellulose acetate (left panel) and nitrocellulose (right panel) membranes empirically so as to yield comparable signal on the nitrocellulose membrane between the GFP^C and GFP^N104 samples, with two-fold dilution in subsequent wells.