Figure 5. KLK6 is constitutively secreted following its overexpression in primary cortical neurons and reduces secreted α-synuclein protein levels.

Adenoviral vectors that drive the expression of stabilized active KLK6 R80Q (Ad-KLK6 R80Q) or inactive KLK6 S197A (Ad-KLK6 S197A) were generated and used to transduce (50 multiplicity of infection) primary neuronal cortical cultures prepared from wt mice (embryonic day 16). 72 hours post-infection cells and CM were collected. (A) Cell lysates (40 μg) and (B) CM concentrated 50-fold were subjected to electrophoresis and analyzed by immunoblotting, using the syn-1 and the KLK6 antibodies. β-actin and BSA were used as loading controls. Protein levels of virally expressed active (R80Q) and inactive (S197A) KLK6 in cell lysates were evaluated by densitometry quantification. Band intensities were normalized versus β-actin. Bars represent the mean ± S.D. of three independent experiments. (C) Detection of active KLK6 using an activity-based probe (ABP) developed in-house (our unpublished data). The ABP carries an organophosphorus ester as a reactive group and a biotin tag. β-actin and BSA were used as loading controls.