Ctd 19

The following antibodies were used in this study: CTSD monoclonal antibody CTD-19 (ab6313; Abcam, Cambridge, MA, USA; 1 : 1000 dilution), β-actin monoclonal antibody AC-15 (A5441; Sigma-Aldrich, St. Louis, MO, USA; 1 : 10 000 dilution), p62 monoclonal antibody p62 LCK ligand (610833; BD Transduction Laboratories, Swampscott, MA, USA; 1 : 1000 dilution), ubiquitin polyclonal antibodies (Dako, Glostrup, Denmark, and Chemicon, Temecula, CA, USA; 1 : 1000 dilution), α-synuclein monoclonal antibody (610787; BD Biosciences, San Diego, CA, USA; 1 : 1500 dilution), α-synuclein monoclonal antibody Ab274 (1 : 1500 dilution), α-synuclein monoclonal antibody Ab62 (1 : 1000 dilution), HRP-conjugated goat anti-mouse IgG (H+L) (172-1011; Bio-Rad Laboratories, Hercules, CA, USA; 1 : 3000 dilution) and HRP-conjugated goat anti-rabbit IgG (H+L) (Bio-Rad Laboratories; 1 : 3,000 dilution).
Fluorescein-conjugated dextran (10 000 molecular weight; D-1821), TO-PRO-3 iodide (T3605) and LysoTracker Red DND-99 (L-7528) were purchased from Invitrogen (Carlsbad, CA, USA).

Cells were fixed in 3.7% paraformaldehyde (pH 7.4), permeabilised with methanol for 10min at -20^°C, blocked in 2% (v/v) goat serum in PBS with 0.1% (v/v) Triton X-100 for 1h at RT, and then incubated with HA antibody (HA.1, 1:1000, Covance) at 37 °C, or ATG16L (D605, 1:100, Cell Signaling), cathepsin D (CTD-19, 1:100, Abcam) and Rab7(EPR7589, 1:100 Abcam) antibodies overnight at 4^°C. Following washing of cover slips with PBS, cells were incubated with anti-mouse or anti-rabbit alexa fluor (Invitrogen) for 1h at 37 °C, washed with PBS and coverslips mounted in citifluor containing DAPI.