Fig. 7.

Quaternary structures and activities of Bri2 BRICHOS secreted from mammalian cells and in serum. a Conditioned culture medium from SH-SY5Y cells was immunoprecipitated for Bri2 BRICHOS and analyzed by SDS-PAGE and Western blot under reducing or non-reducing conditions, and in the presence (+) and absence (−) of N-Ethylmaleimide (NEM). The band at 50 kDa is found after the same treatment of non-conditioned medium. b Culture medium from HEK293 cells transfected with Bri2 BRICHOS was immunoprecipitated for Bri2 BRICHOS and analyzed by SDS-PAGE and Western blot under reducing or non-reducing conditions. c Monomeric rh Bri2 BRICHOS incubated at 4 °C or 37 °C overnight and analyzed by SDS-PAGE under non-reducing conditions. SDS-PAGE of the same samples under reducing conditions is shown in Supplementary Fig. 9c. d Monomeric rh Bri2 BRICHOS incubated in mouse serum at 37 °C, or serum only, analyzed by Western blotting under non-reducing conditions at different time points (in hours) as indicated above each lane. Western blotting of the same samples under reducing conditions is shown in Supplementary Fig. 9d. e Aggregation kinetics of 3 µM Aβ42 alone (black) or with 50% molar ratio of rh Bri2 BRICHOS monomer incubated at 4 °C (red) or at 37 °C overnight (blue). f Aggregation kinetics of 600 nM CS at 45 °C alone (black), in the presence of 1.2 µM rh Bri2 BRICHOS monomer incubated at 4 °C (red) or at 37 °C overnight (blue). g Aggregation kinetics of 600 nM CS at 45 °C in the presence of non-incubated serum (black), serum incubated at 37 °C (yellow), non-incubated rh Bri2 BRICHOS/serum mixture (red), and rh Bri2 BRICHOS/serum mixture incubated for 24 h at 37 °C (blue). The data are presented as means ± standard deviations of 3–4 replicates of experiments that have been repeated three times with qualitatively similar results