4 hydroxynonenal hne

Anti-vimentin antibodies used included mouse monoclonal V9 (Santa Cruz Biotechnology, sc-6260), mouse monoclonal E5 (Santa Cruz Biotechnology, sc-373717), rabbit polyclonal “C-term” (RayBiotech, 102-15110), goat polyclonal “C-end” (Everest Biotech, United Kingdom, EB11207) and “N-term” (Cell Signaling Technology, D21H3), as well as the V9 and E5 conjugates with Alexa Fluor 488 or Alexa Fluor 546, and Alexa Fluor 647 conjugated D21H3, as specified where it corresponds. Anti-actin antibody was from Sigma (A2066). Several secondary antibodies were used: anti-mouse, anti-rabbit and anti-goat immunoglobulins, conjugated with Alexa Fluor 488, 546 or 647, all obtained from Molecular Probes. HRP-conjugated anti-mouse, anti-rabbit and anti-goat antibodies were from Dako. 4,6-diamidino-2-phenylindole (DAPI), Phalloidin-TRITC and acrolein were from Sigma. 4-hydroxynonenal (HNE) was from Cayman Chemical. Calyculin A was from Santa Cruz Biotechnology.

Recombinant α-SYN (140 μm) was produced as described previously (Näsström et al., 2011 (link)). Monomeric α-SYN was incubated with 4-hydroxy-nonenal (HNE) (Cayman Chemicals) in a HNE: α-SYN ratio of 30:1 at 37°C for 72 h. SEC-HPLC was performed on a Superose 6 PC 3.2/30 column (GE Healthcare) and revealed a near complete conversion from monomers to oligomers (Fig. 1-1B). As an eluent, 20 mm Tris and 0.15 m NaCl, pH 7.4, was used at a flow rate of 50 μl/min. The α-SYN oligomers were labeled with the Cy3^AM Antibody Labeling Kit (GE Healthcare, PA33000), the Atto 488 protein labeling kit (Sigma-Aldrich) or pHrodo (Invitrogen, P36600). The pHrodo dye reacts with amine groups on the cell surface and is nonfluorescent at neutral pH, but emits red light at an increasing intensity as the pH is lowered. For all labeling, the protocols provided with the dye kits were followed and unbound excess Cy3, Atto 488, or pHrodo were removed by filtration in a Zeba spin desalting column (Thermo Scientific) according to the manufacturer's instructions.