(A) Size-exclusion chromatography profiles of wild-type HSPB6, Δ31–40 and Δ61–70. 100 µl of protein at three different concentrations was loaded on a Superdex 200 10/300 GL column. 2.5 mg/ml is depicted as a solid line, 5 mg/ml as a dashed line and 10 mg/ml as a dotted line. The absorbance was normalized to the maximum absorbance for each curve. (B) Bis-ANS titration curves for HSPB1, HSPB6 and HSPB4. 2.5 µM protein was mixed with increasing concentrations of bis-ANS and fluorescence intensity was recorded at 490 nm using an excitation wavelength of 390 nm. HSPB6 is shown in yellow, HSPB1 in black and HSPB4 in grey. (C) Bis-ANS titration curves for all constructs, the same setup as for HSPB1 and HSPB4 was used. HSPB6 is shown in yellow, ΔN11 in blue, Δ11–20 in cyan, Δ21–30 in red, Δ31–40 in green, Δ41–50 in orange, Δ51–60 in pink and Δ61–70 in violet.