For NM-HA and NM-HA-C immunocapture, native lysates were prepared as described [61 (link)], and immunocapture was performed using anti-HA magnetic beads or anti-Myc magnetic beads (Thermo Scientific Pierce). Co-captured proteins were resolved by SDS-PAGE and analyzed by western blotting for Sup35, HA (Roche), Hsp104 (Abcam), Ssa1 (gift from E. Craig), and Sis1 (gift from M. Tuite). The amount of Sup35 and NM-HA or NM-HA-C captured was adjusted to reflect only the amount of Sup35 proteins present in aggregates in each strain, as determined by incubating lysates at 53°C and 100°C in the presence of SDS and resolving the protein on an SDS-PAGE gel. The percentage of protein in aggregates was then calculated as the fraction of Sup35 that did not enter the gel at 53°C. The amount of each chaperone that was co-captured was then compared to the amount of captured aggregated Sup35.
For Hsp104 binding to luciferase reporters, cells were incubated at 37°C for 30 minutes followed by 40°C for 35 minutes, with the addition of cycloheximide to 100μg/mL for the last 10 minutes. Cell lysates were prepared, and immunocapture was performed as described [61 (link)], except 600mM NaCl was used in lysis and wash buffers. Co-captured proteins were separated by SDS-PAGE and analyzed by western blotting for Firefly luciferase (Sigma) and Hsp104 (Abcam).
For Hsp104 binding to luciferase reporters, cells were incubated at 37°C for 30 minutes followed by 40°C for 35 minutes, with the addition of cycloheximide to 100μg/mL for the last 10 minutes. Cell lysates were prepared, and immunocapture was performed as described [61 (link)], except 600mM NaCl was used in lysis and wash buffers. Co-captured proteins were separated by SDS-PAGE and analyzed by western blotting for Firefly luciferase (Sigma) and Hsp104 (Abcam).
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