Microson ultrasonic cell disruptor xl

ChIP assay was carried out using a SimpleChIP Plus Sonication Chromatin IP kit (Cell Signaling Technology, Beverly, USA) according to the manufacturer’s protocol. Briefly, cells were fixed with 1% formaldehyde and chromatin was sheared with a Microson XL ultrasonic cell disruptor (Misonix, Farmingdale, USA). Then, 10 μl of solution was used as the input. The surplus samples were incubated with anti-ets1 antibody (Abcam) or IgG for 10 h at 4°C. After the immunoprecipitants bound to protein G magnetic beads, the beads were washed and incubated at 65°C for 2 h. After purification, the sequence of DNA fragments was detected by PCR analysis. The primer sequences for
PTP1B are listed below: forward 5′-CATTATTCAACACACTTCCCA-3′, and reverse 5′-GGACACTTGTGCTATTTTGAG-3′.

RS4;11 cells (30 × 10⁶) were fixed in MEM medium containing 1% formaldehyde for 10 min at room temperature; the reaction was stopped by glycine quenching (125 mM final concentration). Nuclei were collected, digested in 50 mM Tris-HCL pH 8.1, 10 mM EDTA, 10% SDS, and then sonicated (3 cycles, consisting of 30 s with and without sonication) using a Microson XL ultrasonic cell disruptor (Misonix Inc., Farmingdale, NY, USA). Proteins tied to DNA fragments (ranging from 100–600 bp) were pulled-down overnight at 4 °C using appropriate antibodies, then mixed with protein-G magnetic beads (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and incubated for 2 h. Beads were washed with ChIP buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA, 10% SDS, 0.5% EGTA, 140 mM NaCl, 10× Na-deoxycholate, 100× Triton). Immunoprecipitates were dissolved in elution buffer (0.5 M EDTA, 1 M Tris-HCl pH 8.0) and DNA was isolated by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Real time quantitative PCR (RT-qPCR) was performed with 1 µL of DNA using a custom-made primer set [17 (link)].

Uteri were removed from 50 T. trichiura females using a 30G ½” needle (BD Microlance, Fraga, Huesca, Spain) (10x – 30x magnification) and placed in phosphate buffered saline (PBS; pH 7.4). The uteri were opened with a longitudinal incision to facilitate the release of non-embryonated eggs which were pooled. After five washes in PBS (10,000 g; 1 min) the supernatant was removed and a 1% protease inhibitors cocktail (Complete mini EDTA-free, Roche, Berlin, Germany) with 1% Triton X-100 (Sigma-Aldrich, Steinheim, Germany) in PBS added to the egg pellet which was homogenized as described previously [30 (link)]. To ensure disruption of Trichuris eggshells, the homogenate was sonicated while frozen at -20°C using ten cycles of 10x 1-second pulses at maximum intensity with a Microson Ultrasonic Cell Disruptor XL (Misonix, Farmingdale, NY, USA). Homogenates were checked for egg disruption under a stereomicroscope, centrifuged (10,000 g; 10 min at 4°C), and the supernatant containing the soluble NE egg proteins recovered (the T. trichiura NE egg extract—EE). The EE total protein concentration was determined by a commercial Protein Assay (Bio-Rad, Hercules, USA) based on the Bradford method of quantification of soluble proteins [31 (link)] and stored frozen at -20°C until further analysis.