JCS02809 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Alignment of T. brucei MCA2 (Tb06.3A7.310), MCA3 (Tb06.3A7.290) and MCA5 (Tb09.211.4760) was performed using Align X (Invitrogen). Identical residues in the three sequences are shaded black, conservative residues are shaded grey. The putative active site His and Cys residues are indicated above by an asterisk and predicted WW binding domains are indicated below by black lines. The dotted line above shows the region of MCA2/MCA3 chosen for generation of a long peptide for antibody production.

• Supplemental Figure 2 -

  Fig. S2. Localisation of metacaspases. BSF parasites were incubated with several probes to specifically label organellar compartments and co-labelled with anti-MCA5. Early endosomes were labelled with tomato lectin (TL, green), the lysosomal compartment was stained using Lysotracker (Lys, red) and the Golgi apparatus was stained using BODIPY Ceramide (BODIPY, red). Cells were washed in serum-free HMI-9 medium and resuspended in 100 ml of serum-free HMI-9 at 1310⁷ ml^–1. 1 ml of FITC-tomato lectin (1.3 mg ml^–1 stock, Sigma) was added and incubation was in the dark at 4°C for 30 minutes. The cells were then washed and incubated for 30 minutes at 37°C in serum-free pre-warmed HMI-9. Lysosomes were selectively stained using log phase BSF parasites, washed and resuspended in 100 ml of serum-free HMI-9 at 1310⁷ ml^–1. 1 ml of Lysotracker Red DND-99 (1mM stock, Molecular Probes) was added and incubated at 37°C for 20 minutes. Golgi apparatus staining was performed on fixed parasites previously adhered to slides. 1 ml of Bodipy-Texas Red ceramide (1mM stock, Molecular Probes) was added to 1 ml of 1.8% (w/v) fatty acid-free BSA in PBS and the parasites incubated for 1 hour at 4°C. Bars, 10 mm.