Complete mini edta free

In order to obtain saliva, 400 semi-engorged female ticks were taken directly from susceptible hosts (Holstein breed), washed, dried and injected in the haemocoele with a solution of dopamine (Revivan®, Zambon, São Paulo, Brazil). Saliva was collected in protease inhibitors according to manufacturer’s recommendations (Complete, Mini EDTA-free, Roche, Basel, Switzerland) and frozen at -20 °C. FSG and MSG were collected from ticks fed on susceptible (hereafter designated FSGS or MSGS) and resistant hosts (FSGR or MSGR) under a dissecting microscope and with sterile dissection tools and the glands were put in water with protease inhibitors. The samples were sonicated in order to obtain the extract. The extracts of UFL were obtained from 1 mg of egg masses from female ticks fed on resistant or susceptible hosts (hereafter designated UFLS and UFLR). The larvae were put in water with protease inhibitors according to the manufacturer’s recommendations (Complete, Mini EDTA-free, Roche, Basel, Switzerland), followed by pulverization using a pestle and liquid nitrogen in order to obtain the extract. Proteins concentrations were measured by the Coomassie assay, according to the manufacturer’s instructions (Pierce, Rockford, IL, USA).

Cells in 10 cm dishes were transfected at 50% confluency. At 80% confluency cells were washed once with cold PBS, before being scraped, pelleted and lysed in 100 μl PBS, 0.1% (v/v) NP40 by pipetting. Nuclei were recovered by short centrifugation and the supernatant was discarded. After washing once with ice-cold PBS, nuclei were lysed successively in 100 μl strip buffer (10 mM Tris–HCl pH 7.4, 1 mM EGTA, 1.5 mM KCl, 5 mM MgCl₂, 290 mM sucrose, 0.1% (v/v) Triton X-100, 1 mM DTT, 1× cOmplete mini EDTA-free (Roche)), 100 μl low/medium/high salt lysis buffer (20 mM HEPES–KOH pH 7.9, 25% glycerol, 200/400/800 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 1× cOmplete mini EDTA-free (Roche)) for 5 min on ice each. Supernatants were combined and salt concentration was adjusted to 150 mM KCl with lysis buffer without KCl. Extract was cleared at 21 000 × g for 5 min and directly used for pull-down experiments.

Total proteins were extracted from U87-MG cells using RIPA buffer (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; and 0.1% SDS) containing a protease inhibitor cocktail (complete, Mini, EDTA-free, Roche Applied Science, Penzberg, Germany) and homogenized using an ultrasonic homogenizer. After quantitative analysis using the Bradford protein assay with a Bio-Rad Dye Reagent (500-0006, Bio-Rad, Hercules, CA, USA), the protein samples were denatured with 5× sample buffer (10% SDS, 25% beta-mercaptoethanol, 50% glycerol, 0.25 M Tris-HCl [pH 6.8], and 0.01% bromophenol blue) and incubated in a water bath at 95°C for 10 min. Cell protein lysates were loaded into 10% SDS-polyacrylamide gels. The gels were transferred onto PVDF filters and incubated with a specific primary antibody to caspase-3 (1:1000) (Novusbio Inc., USA), cleaved caspase-3 (1:1000) (Novusbio Inc., Centennial, CO, USA), PARP (1: 1000) (Cell Signaling Inc., Danvers, MA USA), cleaved PARP (1:1000) (Cell Signaling Inc.), and β-actin (1:1000) (Cell Signaling Inc.). All blots were normalized to the internal standard of β-actin. Bands of interest were visualized using ECL reagents (PerkinElmer, Waltham, MA, USA) and quantified using the UVP BioImaging system (Biospectrum AC Imaging System, Upland, CA, USA) and the ImageJ software program (National Institutes of Health, Bethesda, MD, USA).