Monoclonal anti v5

For immunofluorescence 1 × 10⁸ of BF trypanosomes were washed with PBS supplemented with 10 g/l glucose, and spread onto slides coated with polylysine (100 μg/ml; Sigma). The cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), washed with PBS, and permeabilized with 0.1% triton X-100 in PBS. The cells were then washed with 1xPBS-T (0.05% Tween). After blocking with 5.5% FBS, the respective slides were incubated for 1 hr in PBS-T plus 3% BSA with the following primary antibodies: monoclonal anti-v5 (1:200, Invitrogen), anti-enolase (1:400, [36 (link)]) and anti-hexokinase (1:400, a gift from Paul Michels). After washes, the slides were incubated in the dark for 1 hr with the following secondary antibodies: goat anti-rabbit IgG (H + L) Texas Red conjugate (1:400, Life Technologies) or goat anti-mouse IgG (H + L) FITC conjugate (1:400, Sigma). The slides were washed and mounted in Vectashield containing 1.5 μg/ml DAPI (Life Technologies).

Expression of the target protein was compared between uninduced and induced RNAi cell cultures using cell lysates corresponding to 5 x 10⁶ cells per lane. Lysates were prepared in NuPAGE^® LDS sample buffer (Invitrogen), separated on Bolt 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to an Amersham Hybond P PVDF membrane (GE Healthcare) for probing with the respective antibodies. Rat anti-alpha MPP, and rabbit anti-MIP were used at dilutions 1:1,000, 1:1,000 and 1:5,000, respectively. Polyclonal anti-enolase [48 (link)] and anti-trCOIV antibodies [49 (link)] were used at dilutions 1:50,000 and 1:10,000, respectively. Monoclonal anti-mtHsp70 was used at a dilution of 1:5000. Monoclonal anti-V5 (Invitrogen), polyclonal anti-GFP (Life Technologies) and monoclonal anti-tubulin antibodies (Sigma-Aldrich) were used at dilutions 1:2,500, 1:2,000 and 1:5000, respectively. Secondary antibodies were conjugated to horseradish peroxidase (Sigma-Aldrich), and the signal was visualized using Clarity Western ECL Blotting Substrate (Bio-Rad).