Figure 6. Hsp104 disaggregates ubiquitinated Q394X.
(A) A schematic of the flotation assay used to measure substrate oligomerization. Cell lysates from HSP104 or hsp104Δ strains expressing the Q394X mutant incubated at 37°C for 1.5 hrs were prepared and analyzed by equilibrium sucrose density centrifugation. Proteins in each fraction were TCA precipitated and immunoblotted with anti-HA antibody to detect (B) Q394X, or (C) were denatured and immunoprecipitated to detect ubiquitinated Q394X. For (C), compare the boxed regions in lanes 6 and 7 from wild type and hsp104Δ cells, as well as the quantification to the right. (D) The migration of Sec61 was used to mark the ER. Data represent the means of N = 4–5 independent experiments, +/− SEM; * P < 0.05.