Hsp110, Hsp70 and Hsp40 are key components of the mammalian disaggregation machinery.

<p>(<b>A, B</b>) Immunoblots showing depletion of either p97, Hsp70, Hsc70, Hdj1, Hdj2, Hsp105 or Apg-2 from either RLC (<b>A</b>) or SHC (<b>B</b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026319#s3" target="_blank">Materials and Methods</a>. 20 µg of cytosol was fractionated by SDS-PAGE and processed for immunoblot. (<b>C–F</b>) Urea-denatured luciferase aggregates (50 nM) (<b>C, D</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>E, F</b>) were incubated for 6 h in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated cytosol (10 mg/ml), mock-depleted cytosol (10 mg/ml) or depleted cytosol (10 mg/ml). In the indicated reactions, the small molecule p97 inhibitor, DBeQ (100 µM), or Hsp70 inhibitor, VER 155008 (100 µM), were included. In the indicated reactions, Hsc70-depleted cytosol was supplemented with Hsc70, Hdj1-depleted cytosol was supplemented with Hdj1 and Apg-2-depleted cytosol was supplemented with Apg-2 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026319#s3" target="_blank">Materials and Methods</a>. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (<b>C, D</b>). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (<b>E, F</b>). Values represent means ± SEM (n = 3).