Irdye streptavidin

Parasites were incubated with or without 10 μM of the inhibitor 1, 5, or 8 in 1 mL 2% delipidated BSA-DMEM at 37°C for ICA and TCT and at 28°C for Epi. After 6 h, 100 μM myristic acid, azide (from a 50-mM stock in DMSO) were added to the samples, and the same volume of DMSO was added to the negative control. Cells were incubated for another 6 h, for a 12-h treatment with the inhibitors. Next, the parasites were washed three times with PBS and lysed in 1% SDS, Tris-HCl, pH 8.0. “Click” reaction was performed between the myristic acid azide and Biotin Alkyne (Invitrogen) according to the manufacturer instructions using the Click-iT Protein Reaction Buffer Kit (Invitrogen). Duplicate aliquots of lysates were ran in 10% SDS-PAGE followed by fixing (5% acetic acid, 50% isopropanol) for 15 min. Gels were then analyzed by in-gel western blotting using IRDye streptavidin (LI-COR Biosciences, Lincoln, NE) 1:7500 in 5% BSA, 0.05% SDS, 0.2% Tween in PBS, for 1 h at room temperature. They were subsequently treated or not with 0.2 M NaOH in methanol for 1 h at room temperature. Gels were scanned in an ODYSSEY quantitative imagining system (LI-COR Biosciences). They were next stained with Coomassie blue and scanned again to determine total protein levels.

Whole cell lysates prepared directly in SDS-PAGE sample buffer, or membrane-enriched fractions prepared by solubilising digitonin-permeabilised cells in sample buffer, were resolved by SDS-PAGE and then transferred onto PVDF membranes (Immobilon, IPFL00010) at 300 mA for 140 min in transfer buffer [25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.25% (w/v) SDS, 20% (v/v) methanol]. Membranes were incubated in 10% (v/v) blocking buffer (Sigma-Aldrich, B6429) in Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.6, 150 mM NaCl) for 1 h at room temperature. Primary antibodies diluted (1:1000) in 10% blocking buffer/TBS-T [TBS with 0.1% (w/v) Tween] were added to membranes overnight at 4°C. Membranes were washed with TBS-T and appropriate secondary antibodies diluted in 10% blocking buffer, TBS-T with 0.01% SDS were added for 1 h. Membranes were washed and scanned using the Odyssey Infrared imaging system (LI-COR Biosciences, 700 nm and 800 nm, 169 μm resolution). Dye-conjugated IRDye secondary antibodies (rabbit or mouse, 680/800 nm; LI-COR) and streptavidin IRDye (LI-COR Biosciences) were used for quantitative immunoblotting (1:10,000).