Protease inhibitor

The protein extraction was performed with cells grown in the presence and absence of argentilactone. After incubation with 9 μg/mL argentilactone for 6 h in MMcM liquid medium, the cells were centrifuged at 10,000 x g for 15 min at 4°C and protein was extracted using extraction buffer (20 mM Tris-HCl pH 8.8; 2 mM CaCl₂) containing a mixture of protease inhibitors (GE Healthcare). After the addition of glass beads (0.45 mm), the cells were lysed in a bead-beater, followed by centrifugation at 10,000 x g for 15 min at 4°C. The supernatant was collected, and the protein concentrations were determined using Bradford reagent (Sigma-Aldrich) [27 ].

Purified BED was dialyzed overnight at 4 ˚C against 25 mM HEPES.Na pH 7.5, 150 mM NaCl, 0.3 mM TCEP, and protease inhibitors (Roche) and subsequently concentrated (Vivaspin 20 3 kDa MWCO, GE Healthcare) to ~7 mg/mL. The samples (60 µL) were prepared by mixing the protein and the oligonucleotide from the 500 µM stock solution in various ratios (1:0, 0:1, 1:1, 2:1, and 3:1 DNA-to-protein ratio with 1 equivalent corresponding to 100 µM) and equilibrated on ice for at least 15 min. About 10 µL of the sample (10 µl injection loop loaded with 50 µL sample) were injected on a Superdex 75 PC 3.2/30 (GE Healthcare) analytical size exclusion column preequilibrated with the protein buffer. The UV absorbance at 280 and 260 nm were monitored to identify the different species.

Protein extracts were obtained after 8 h incubation in MMcM in the presence of 79 μM TSC-C or in its absence. Yeast cells were centrifuged at 10,000 x g for 10 min at 4°C, and the proteins were extracted using extraction buffer (20 mM Tris-HCl pH 8.8; 2 mM CaCl₂) with a mixture of protease inhibitors (GE Healthcare). After the addition of glass beads (0.45 mm), the cells were lysed in a bead-beater, followed by centrifugation at 10,000 x g for 15 min at 4°C. The supernatant was collected and used in enzyme activity assays. The protein concentrations were determined using the Bradford reagent (Sigma-Aldrich), as previously described [37 ].

The placenta and endometrium from 18 IUGR and 18 NBW (6/stage) were used to extract protein, as we described previously [3 (link)]. Briefly, approximately 0.2 g frozen samples were crushed to powder in liquid nitrogen, then homogenized in a lysis buffer containing 7 M urea, 2 M thiourea, 4% 3-[3-(-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 50 mM dithiothreitol with protease inhibitors (GE Healthcare, Piscataway, NJ). An ultrasonicater (Sonics Model VC 750, Sonics and Materials, Newtown, CT) was set at 20% power output and used to break down the mixture for 10 min at 0°C. After the addition of 1% (vol/vol) nuclease mix (GE Healthcare), the mixture solution was kept at room temperature for 1 h to completely solubilize proteins, followed by re-sonification for 10 min as described above to thoroughly break down cell membranes. The homogenate was centrifuged for 10 min at 13,000 g at 4°C to settle down the insoluble components. The supernatant fluid was obtained and its protein concentration was determined using the Brandford method. Portions of the homogenate (1 mg of protein for both placenta and endometrium) were stored at -80°C.

Intoxication treatment for proteomic analysis was performed by treating two-day-old A. aegypti larvae with the Ag/AgCl NPs at the LC₂₅ concentration. After 24 h treatment, the surviving larvae were cultured further for 24 h without Ag/AgCl NPs. The midgut of the surviving A. aegypti larvae was dissected. A. aegypti larvae without NP treatment were used as a negative control. Samples of 100 midguts were pooled in 100 µL of rehydration solution (8 M urea, 2% w/v CHAPS, 40 mM dithiothreitol, 0.5% v/v IPG buffer pH 3–10 (GE Healthcare Life Sciences, Uppsala, Sweden), 0.02% w/v bromophenol blue) containing protease inhibitors (Roche Diagnostics, Mannheim, Germany). Then, the midguts were homogenized on ice using a pellet pestle (Sigma-Aldrich, Saint Louis, MO, USA) and the extracted proteins were cleaned using a 2-D Clean-Up kit (GE Healthcare Life Sciences, Uppsala, Sweden), following the manufacturer’s instructions. Then, the protein pellets were solubilized in the rehydration solution and quantified using a 2-D Quant Kit (GE Healthcare Life Sciences, Uppsala, Sweden). The midgut protein concentration was adjusted to 50 μg in 120 μL of the rehydration solution.