Kimble kontes pellet pestle

Only partially engorged females of H. dromedarii were used for DNA extraction where five ticks from each month were randomly selected. Before DNA extraction, ticks were washed by using ethanol and distilled water following a protocol (37 (link)). Each selected individual tick was homogenized using a sterile Kimble Kontes pellet pestle (Thermo Fisher, Waltham, MA) in a sterile 1.5-ml microcentrifuge tube. Genomic DNA was extracted from individual ticks using QIAamp Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. A spectrophotometer (Nano Drop ND-1000, Erlangen, Germany) was used to measure the concentration and quality of DNA. In addition, the quality of DNA was checked on 1.5% agarose gel. Genomic DNAs from five ticks were pooled and a total of 12 pools were prepared (one pool for each month) and then the DNA was stored at −80°C in the freezer until further use.

Only male H. anatolicum were selected for DNA extraction due to an insufficient number of female ticks. Before DNA extraction, ticks were washed in 70% ethanol and deionized water for 5 min to remove environmental contaminants in accordance with a published protocol [23 (link)]. Due to the small size of each tick, a pool of five male H. anatolicum ticks was homogenized in liquid nitrogen inside a sterile 1.5-ml microcentrifuge tube using a sterile Kimble Kontes pellet pestle (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was extracted from each pool using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration and quality of DNA were assessed using a Nano Drop ND-100 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). DNA quality was also determined by electrophoresis in a 1% agarose gel; the bands were stained with ethidium bromide and visualized under UV light. DNA was stored at − 20 °C in the freezer until further use. Prior to sequencing, extracted DNA samples (5 DNA samples of ticks from each host) were pooled again to make one pool for each host from each location, resulting in seven DNA pools.

Subcellular fractionation was performed by differential centrifugation in isotonic sucrose buffer with minor modification to previously described protocols [36 (link)]. Briefly, 1 × 10⁸ pelleted cells were resuspended in 1 ml of ice-cold sucrose buffer (0.25 M sucrose in 10 mM Tris-HCl pH 7.4 and Protease Inhibitor Cocktail—Roche Diagnostic) and homogenized for 4 min (10 s ON–10 s OFF) using Kimble Kontes Pellet Pestle (Thermo Fisher Scientific). Homogenized cells were centrifuged at 4 °C at increasing speed to isolate specific cellular compartments, as schematized in Suppl. Figure 5. Pellets were resuspended in sucrose buffer and proteins were quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty-five micrograms of protein extracts for each fraction were then solubilized with 3× Laemmli Sample Buffer with 0.1 M DTT, denaturated and analyzed by western blot using 7.5% or 4–15% polyacrylamide gels (BioRad, Munich, Germany). LAMP2, Pan-cadherin, GAPDH, VDAC, Histone H3, and β-tubulin were used as specific markers for lysosomes, plasma membranes, cytosol, mitochondria, nuclei, and total fractions, respectively.