Figure 7. Klk6^–/– primary neuronal cultures internalize exogenously added fibrilar forms of α-synuclein with higher efficiency compared to wt cultures.

Primary neuronal cortical cultures were prepared from wt or Klk6^–/– mice and were cultivated for 9 days in vitro. (A) Neuronal cultures were treated with human recombinant forms of α-synuclein (monomer, fibrils and ribbons) for 4 days and (B) with mouse or human fibrilar forms of α-synuclein for 3 and 4 days. Neurons were lysed in 1% TX-100 and internalized α-synuclein was detected by immunoblotting, using specific antibodies against either the human-specific (4B12) or total (human and rodent) α-synuclein (C-20)-R. (C) To detect insoluble α-synuclein species, lysates were further extracted in 1% sarcosyl and α-synuclein levels were detected using the (C-20)-R antibody. β-actin was used as loading control. Arrows indicate monomeric α-synuclein, brackets indicate α-synuclein higher molecular weight species.