Figure 3

HSPB1-3D is required for aggregate resolubilization. (a, b) SEC analysis of native (a) luciferase and (b) LDH. After chromatography, the elution patterns of proteins were detected by immunoblot (IB). For comparison, 10% of total protein (10%T) before chromatography was also detected. (c–f) Disaggregation of aggregates composed of (c) luciferase and HSPB1, (d) LDH and HSPB1, (e) luciferase and HSPB1-3D, or (f) LDH and HSPB1-3D. The aggregates were formed at 45 ℃ or 55 ℃ for 15 min and transferred to disaggregation reactions containing buffer (− Chap) or the chaperone disaggregation machinery (+ Chap) and ATP. Aggregate resolubilization at the start (0 h) and end (3 h) of the disaggregation reactions was analyzed by SEC and immunoblot (IB). (g, h) Quantification of (g) luciferase or (h) LDH aggregates with HSPB1-3D from e and f in eluted fractions are shown as percentage of the total amount of immunoblot signal. Error bars show standard deviations, n ≥ 3.