Branson sonifier 250

Bacterial suspensions of recombinant E. coli used to overexpress proteins were lysed by ultrasonic vibration with a Branson sonifier 250 (Branson Ultrasonics, Brookfield, CT, USA), using 6 cycles of sonication of 30 s each with a 40% duty cycle, adding 2% (v/v) Triton X-100 before the last two sonication cycles. Centrifugation at 12,000× g for 30 min at 4 °C was then performed to remove non-soluble cell debris. The cleared supernatants were collected and kept at 4 °C. The BCAL2645 and BCAL2022 6× His-tagged proteins were then purified by affinity chromatography using HisTrap FF columns (GE Healthcare, Chicago, IL, USA). The columns were initially equilibrated by flowing 10 mL of Buffer A (20 mM sodium phosphate, 750 mM NaCl, 20 mM imidazole, 10% glycerol, pH 7.4) for BCAL2645 or Buffer B (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4) for BCAL2022. The proteins were then eluted with 5 mL of Buffer A or B, respectively, containing imidazole concentrations of 60, 100, 150, 200, 250, 300, 400 and 500 mM. Aliquots of 1 mL were collected for each protein, followed by their analysis by SDS-PAGE. Immunoblotting experiments were carried out as previously described using a commercial monoclonal anti-polyhistidine peroxidase conjugate clone HIS-1 antibody (Sigma; St. Louis, MO, USA) [26 (link)]. BCAL2958 purification was performed as previously described [26 (link)].

To evaluate transcription factor binding to key gonadotrope gene promoters, we performed ChIP assays. ChIP assays were performed as previously described [38 (link)]. Chromatin was sonicated to an average length of 300–500 bp using a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). Antibodies recognizing specific histone modifications were: anti-acetyl-Histone-H3 (06–599; Millipore, Temecula, CA), anti-trimethyl-Histone H3-Lys4 (07–473; Millipore), anti-dimethyl-Histone H3-Lys9 (ab1220; Abcam), all of which are ChIP-grade antibodies. To recognize phosphorylated polymerase, ChIP-grade anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131; Abcam, Cambridge, MA), was used. Immunoprecipitated DNA and DNA from input chromatin were analyzed for sequences of interest by qRT-PCR using promoter-spanning primers specified in Table 1. For ChIP assays comparing αT1–1, αT3–1, and LβT2 chromatin, the percentage of enrichment of antibody signal over IgG was calculated for each primer set. IgG was the same species and source as the comparison antibody. Then, values for activating chromatin marks were normalized to those for the inactive gene Gnrh1. For repressive chromatin marks, values were normalized to the highly active gene Actb [37 (link)–39 (link)].

Human recombinant 5-LO was expressed
in Escherichia
coli (BL21, DE3) transformed with the pT3-5-LO plasmid
at 30 °C overnight, as described before.^{26 (link)} Cells were lysed using lysis buffer and homogenized by sonification
(3 × 15 s, Branson Sonifier 250, Branson Ultrasonics Corporation).
5-LO was then purified from 40,000g supernatant (20
min, 4 °C) using an ATP agarose column (Sigma-Aldrich), diluted
with PBS (Dulbecco’s formula, pH 7.4) buffer containing 1 mM
EDTA, and immediately used for 5-LO activity assays.

C. pecorum 1710S (isolate from a swine abortion) was kindly provided by Professor Storz, Baton Rouge, LA, USA [3 (link)] and propagated in HeLa cells. Crude C. pecorum stock was generated by mechanical disruption (scraping) of infected HeLa cells into infection medium, sonication (Branson Sonifier 250; Branson Ultrasonics, Danbury, CT, USA) on ice, centrifugation of infectious particles from the medium at 10,000 g at 4°C for 45 minutes, and suspension in SPG medium. Stock was stored at −80°C and frozen stock aliquots were thawed immediately before infections were carried out. SPG medium consisted of 218 mM sucrose (Sigma-Aldrich), 3.76 mM KH₂PO₄ (Sigma-Aldrich), 7.1 mM K₂HPO₄ (Merck Eurolab AG, Dietlikon, Switzerland), and 5 mM GlutaMAX-100 (GIBCO).

Human blood was taken from a healthy, voluntary donor with a sterile lancet and centrifuged at 14,000×g, for 30 min. The serum was stored at −20 °C. According to the manufacturer’s operating procedure, 10 μL of serum was incubated in a Pierce Top 12 Abundant Protein Depletion Spin Column (Thermo Fisher Scientific, Waltham, USA) for 1 h to deplete high-abundance proteins. The filtrate was concentrated and its buffer exchanged to binding buffer using 10 kDa Millipore Microcon centrifugal filters (Merck Millipore, Darmstadt, Germany) with an Ultracel regenerated cellulose membrane.
In the case of T. atroviride, frozen mycelia was equilibrated to ambient temperature before analysis. Of wet cell mycelia, 100 mg was suspended in 1.6 mL binding buffer and lysed by sonication (intensity 60%, 2× 20 s and 1× 30 s, 1 min waiting intervals each, at 4 °C) with a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT, USA). Lysed cells were centrifuged (14,000×g, 4 °C, 20 min), the supernatant was collected and concentrated by means of 3 kDa Millipore Microcon centrifugal filters.
Protein concentrations of all samples were determined using Bradford Assay and BSA for calibration.

The frozen PBMC pellets were resuspended in 50 mmol/L HEPES lysis buffer (pH 7.4) with 0.3% SDS and protease-inhibitor cocktail tablets (Roche Diagnostic GmbH, Mannheim, Germany). The protein suspension was treated with ultrasonication (Branson Sonifier 250, Branson ultrasonics corp, Brookfield, CT, USA) on ice water, at output control 3 and 30% duty cycle for 3 rounds of 3 pulses with 1 minute on ice between each round. Insoluble proteins were removed by centrifugation at 16,000× g for 10 min at 4 °C.

Fibroblasts were lysed in 100 mM ammonium bicarbonate and 1 M urea with ultrasonication (Branson Sonifier 250, Branson Ultrasonics Corp) at output control 3 and 30 % duty cycle for three cycles of five pulses, with 1 min on ice between each cycle. Lysates were centrifuged at 13,000 g for 30 min at 4°C. Protein concentration of the soluble fraction was measured by Bradford Protein assay (Bio-Rad) and 30 μg were used for SRM analysis. Relative quantification of peptides was carried out using a modified version of the SRM assay described in Fernández-Guerra et al. (2014 (link)). In brief all targeted proteins were monitored by detection of 2–5 tryptic peptides. Defined amounts of heavy labeled synthetic peptide analogs were spiked into the samples and used for relative quantification. The summed fragment ion peak areas for each peptide were normalized to the signal responses from the corresponding spiked heavy-labeled peptide standards. The means of the ratios for each peptide measured in control fibroblasts were set to 100% and the means for the patient fibroblast samples were expressed as the percentage of these. Samples from 3 independently grown control fibroblasts and 3 parallel cultures of patient fibroblasts were analyzed.