Hrp conjugated secondary anti rabbit and anti mouse antibodies

For analysis of protein abundance in the cortex, tissue (Cohort B animals) was dissected under ice-cold PBS before snap freezing. Samples were then homogenised in RIPA Buffer (150 mM sodium chloride, 50 mM Tris, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) plus complete protease inhibitors (Calbiochem) by mechanical disruption. Total protein content was determined by Bradford assay. Samples from individual animals were run separately and were not pooled. Equal amounts of total brain proteins were then denatured in LDS denaturing buffer (Invitrogen) and β-mercaptoethanol, prior to separation by SDS-PAGE gel electrophoresis using precast 4–12% Bis–Tris gels (Invitrogen). Proteins were transferred to nitrocellulose membranes prior to blocking in 5% milk/PBST (0.05% Tween-20). Primary antibodies were diluted in 1% BSA/PBST, HRP conjugated secondary anti-rabbit and anti-mouse antibodies (Dako) were diluted 1:5000 in 1% BSA/PBST. Primary antibodies against SERF2 (Proteintech 11691-1-AP, 1:1000) and β-actin (Sigma A5441, 1:60,000) were used.

To prepare protein samples for SDS‐PAGE trypanosomes were pelleted by centrifugation, washed two times with PBS, resuspended in 1× SDS sample buffer (100 mM Tris–HCl, 4% SDS, 20% glycerol, 0.05% bromophenol blue, pH 6.8) and then heated at 55°C for 10 min. Samples (generally 3–5 × 10⁶ cell equivalents) were separated by SDS‐PAGE on 12% polyacrylamide gels under reducing conditions and transferred to nitrocellulose membrane. Membranes were blocked with 5% milk in TBS, and primary and secondary antibodies diluted in 1% milk in TBS + 0.1% Tween‐20 (TBST). Washing of the membranes after antibody incubations were carried out with TBST. The following primary antibodies were used: anti‐EP247 (1:1,500), anti‐GPEET K1 (1:2,500), anti‐GPEET 5H3 (1:5,000), and anti‐aldolase (a gift from Paul Michels, University of Edinburgh, Scotland, 1:180,000). HRP‐conjugated secondary anti‐rabbit and anti‐mouse antibodies (DAKO) were used at 1:1,000 and 1:5,000, respectively, and chemiluminescent detection was carried out using SuperSignal™ Pico West PLUS substrate (ThermoScientific).

T. brucei cells were harvested by centrifugation, washed twice with PBS and the pellet suspended directly in SDS sample buffer and heated at 55 °C for 10 min. Approximately 5 × 10⁶ cell equivalents per sample were separated by SDS-PAGE under reducing conditions on a 12% polyacrylamide gel and transferred to nitrocellulose membrane. For all blots, blocking was carried out in 5% milk in TBS, and primary and secondary antibodies diluted in 1% milk in TBS + 0.1% Tween-20 (TBST), with the addition of 0.01% SDS for the secondary antibodies. The following primary antibodies were used: anti-p67 (J. Bangs, 1:5000), anti-ALBA1 (I. Roditi, 1:2500), anti-EP247 (Cedarlane, 1:1500), anti-GPEET K1 (I. Roditi, 1:2500), anti-GPEET 5H3 (I. Roditi, 1:5000) and anti-aldolase (P. Michels, 1:180,000). HRP-conjugated secondary anti-rabbit and anti-mouse antibodies (DAKO) were used at 1:1000 and 1:5000 respectively, and chemiluminescent detection carried out using SuperSignal™ Pico West PLUS substrate (ThermoScientific).