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. 2013 Nov 10;19(14):1662–1675. doi: 10.1089/ars.2012.5032

FIG. 4.

FIG. 4.

CSF Frx activity is concentrated in the <3-kDa fraction. (A) CSF from ND cases was fractionated through the filters with different molecular-mass cutoffs. Unfractionated CSF (whole) and purified Cp were processed in parallel. Almost all Frx activity of Cp is detected in the retentate of the 100-kDa, 50-kDa, and 3-kDa filters (>100, >50, and >3) as expected. In contrast, most of the activity from CSF is detected in the <3-kDa filtrate (<3). Data are means±SEM of six CSF samples analyzed in triplicate. (B) CSF was mixed with purified Cp and passed through a 50-kDa cutoff filter. Resulting filtrates were passed through the 3-kDa cutoff filter, and Frx activity was checked in all fractions. Unfractionated CSF and purified Cp were included for internal reference as in (A). Addition of Cp doubles the Frx activity in the CSF+Cp sample, half of which is lost in the >50-kDa fraction (>50). Retentate of the 3-kDa cutoff filter (50-3 kDa) shows no activity, which is detected in the <3-kDa filtrate (<3). Data represent means±SEM of five CSF samples analyzed in triplicate. (C) Purified Cp, a mix of CSF+Cp, and unfractionated CSF were treated with 50 μg/ml of PK at 37°C for 1 h. Control samples received an equivalent volume of PBS. Frx activity of purified Cp is destroyed completely by PK. The CSF+Cp samples show a significant reduction after PK, while CSF samples show no effect of PK treatment. Data represent means±SEM of three samples analyzed in triplicate. Untreated vs. PK-treated, *p<0.05, ***p<0.001. (D) As observed for the ND samples in (A) and (B) above, almost all Frx activity in DM and CJD samples is detected in the <3-kDa fraction (<3), while Cp-associated Frx activity is present in the >3-kDa fraction as expected (>3). Unfractionated CSF and Cp (whole) were analyzed in parallel for internal reference. Frx activity in the CSF of CJD cases is 2.8-fold higher than DM samples. (DM samples include two each of the randomly selected FTD, EN, and AD cases). Data are means±SEM of six cases each of DM and CJD analyzed in triplicate. PK, proteinase-K.