Fig. 2
From: The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro
The recombinant sHSPB5-Fc exhibits chaperone activity in vitro. A, sHSPB5-Fc inhibits heat-induced aggregation of CS in a dose-dependent manner. CS alone or combined with sHSPB5-Fc at a series of molar ratios were incubated at 43 °C in pH 7.4, 40 mM HEPES for 1 h. The absorbance at 320 nm was measured using a microplate reader of multi-wavelength measurement system (Thermo Fisher Scientific). The protection (%) = (1-A320CS + sHSPB5-Fc/A320CS) × 100%. B, Time course to assess the chaperone activity of sHSPB5-Fc to substrate CS. C, Chaperone activity of sHSPB5-Fc on heat-induced aggregation of CS protein using the soluble and insoluble fraction assay. The soluble fraction was separated from the insoluble fraction by centrifugation. The soluble and insoluble proteins were detected with Coomassie blue staining. S, soluble protein. P, pellet of aggregated protein. D, Quantification of pellet versus soluble protein ratio in (C). The densitometry of CS and HSPB5-Fc proteins in the soluble fraction and pellet were measured using Image J software. The gray value ratios of pellet versus soluble protein are shown in the bar graph. Column 1: the ratio of CS alone at 4 °C. Column 2: the ratio of CS alone at 43 °C. Column 3: the ratio of CS when combined with HSPB5-Fc at 43 °C. Column 4: the ratio of HSPB5-Fc alone at 43 °C. Column 5: the ratio of HSPB5-Fc when combined with CS at 43 °C