Pharosfx

DPAP3 activity was measured either using the DPAP fluorogenic substrates VR-ACC[32 (link)] or FR-βNA (Sigma), or with the FY01 activity-based probe. When using FY01, samples (intact parasites, parasite lysates, insect cell supernatant, or rDPAP3 purification fractions) were labelled with 1 μM FY01 for 1 h, boiled in loading buffer, run on a SDS-PAGE gel, and the fluorescence signal measured on a PharosFX (Biorad) flatbed fluorescence scanner [18 (link)]. To determine the potency and specificity of inhibitors against DPAPs and the falcipains, parasite lysates diluted in acetate buffer (50 mM sodium acetate, 5 mM MgCl₂, 5 mM DTT, pH 5.5) were pretreated for 30 min with a dose response of inhibitor followed by FY01 labelling.
When using VR-ACC (10 μM) or FR-βNA (100 μM), substrate turnover was measured on a M5e Spectramax plate reader (λ_Bex = 355 nm/λ_em = 460 nm or λ_ex = 315 nm/λ_em = 430 nm, respectively) in 50 mM sodium acetate, 20 mM NaCl, 5 mM DTT, and 5 mM MgCl₂, pH5.5. The pH dependence of rDPAP3 was determined at 10 μM VR-ACC using a 20 mM sodium acetate, 20 mM MES and 40 mM TRIS triple buffer system containing 5 mM DTT, 0.1% CHAPS, 20 mM NaCl, and 5 mM MgCl₂.

Parasite lysates were diluted to 2 mg/mL in PBSa before labeling with 0.1 to 1 μM of FP-TMR (Thermo Fisher Scientific) for 30 to 60 min. Labeling reactions were quenched by addition of 4X Laemmli buffer (LB, 10% glycerol, 2% SDS, 67.5 mM Tris-HCl, 0.005% bromophenol blue, and 400 mM dithiothreitol (DTT) and heating samples at 95°C for 5 min. After SDS-PAGE of the samples, in-gel fluorescence was measured using a Bio-Rad PharosFX fluorescence scanner (552 nm excitation laser, 572 nm emission filter). In competition assays, parasite lysates were pretreated with HsSH inhibitors (0.2 μM ML348 or 17 μM AA74-1) for 30 min before adding 1 μM or 50 nM FP-TMR for 10 min.

Pre-steady-state burst experiments were performed using an RQF-3 rapid quench-flow apparatus (KinTek Corp.) to determine the active concentration of the purified RT enzymes. The T/P was prepared as above and consisted of a 48-mer DNA template (5′-CGAGCTAAGCGCTTGACCGCAGAACATTGCTAGCGGGAATTCGGCGCG-3′) and a 21-mer primer (5′-CGCGCCGAATTCCCGCTAGCA-3′, template:primer ratio of 2.5:1). In this experiment, 300 μm dATP and 10 mm MgCl₂ were rapidly mixed with RT (100 nm total protein) prebound to T/P (300 nm). All concentrations represent the final concentrations after mixing. The reactions were quenched at various time points with 0.3 m (final) EDTA. Products were then separated on a 20% polyacrylamide/8 m urea gel, visualized using a PharosFX (Bio-Rad), and quantified with Molecular Imager FX software (Bio-Rad). Product formation was fit to the burst equation
in which A is the amplitude of the burst, k_obs is the observed first-order burst rate constant, and k_ss is the linear steady-state rate constant (23 (link)).

Proteins obtained from lysate tissue after 24 and 48 h were size fractioned in 4–12% SDS-polyacrylamide gel, before being transferred to PVDF membrane (GE Healthcare, U.K.) by using Trans-Blot Turbo System (Bio-Rad, U.S.A.). Membranes were blocked for 2 h with 5% bovine serum albumine (BSA) in transfer buffer saline (TBS: 2.5% Tris-HCl, 8% NaCl, 0.1% Tween 20, pH 7.4) at room temperature. Afterward the membranes were incubated in 3% TBS–BSA with primary antibodies overnight at 4°C (Supplementary Table S2).
After two washes in PBS 1×, the membranes were incubated with the secondary conjugated antibodies antimouse Cy3 or antirabbit Cy5 (GE, U.S.A.) diluted 1:2500 in 3% TBS-BSA for 2 h (Supplementary Table S2). Immunolabeling was visualized using the ECL Plex procedure (GE) and the laser scanner Pharos FX (Bio-Rad). Bands were quantitated using densitometric image analysis software (Bio-Rad) or ImageJ software. Molecular mass was determined using a wide range protein marker 8–200 kDa (Bio-Rad). Protein loading was controlled by antiactin (Sigma) detection and was statistically evaluated.

Cy3-labeled anti-mouse IgG antibody was dissolved and diluted in phosphate-buffered saline (PBS, pH 7.4) to obtain 1.6 g/mL solution. Then each 100 L of the solution was applied to three wells of a multilayered microplate with a 200-nm-thick silver layer and 53-nm-thick PPF, and of an unmodified one. Immediately a two-dimensional Cy3 fluorescence image of microplates was obtained using 532 nm excitation with a bandpass filter of 605 nm of a scanner (Pharos FX, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Fluorescence intensity was quantified as follows: an empty circle equivalent to the same area of a well of microplate was created on the scanned image by Quantity One Software (Bio-Rad). Circles were copied and overlaid on all the wells of interests, and fluorescence intensities within the circle area were calculated by subtracting the background signals from wells containing only buffers on each type of microplate.