Kanamycin

BL21(DE3) cells harboring pCOLADuet-1, pCOLADuet-1_AtDTX1, or pCOLADuet-1_NtJAT1 were cultured overnight in liquid LB medium containing 50 mg/L kanamycin (Nacalai Tesque) at 30 °C with shaking at 200 rpm. Overnight cultures of OD₆₀₀ = 0.1 were then inoculated in 90 mL LB medium containing 50 mg/L kanamycin and grown at 30 °C. Isopropyl β-D-thiogalactopyranoside (IPTG) (1 mM) was added when OD₆₀₀ reached 0.6, then were incubated for another 3 h. Next, the cells were harvested via centrifugation at 9000×g for 2 min, re-suspended in 0.5 mL 1 × phosphate-buffered saline (PBS), and disrupted via sonication (nine cycles of 30 s on, 30 s off, performed on ice). The mixture was centrifuged at 3000×g for 15 min, and the supernatant was collected and centrifuged at 20,000×g for 20 min to pellet the membrane proteins. The membrane proteins were then denatured, subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Tokyo, Japan). The membrane was treated with BlockingOne (Nacalai Tesque) and incubated with an anti-His monoclonal antibody (MBL Life Science, Nagoya, Japan) against His-AtDTX1 or an anti-NtJAT1 antibody (a generous gift from Dr. Y. Moriyama, Kurume University) against NtJAT1. The immunoreactive band was visualized using Chemi-Lumi One Super (Nacalai Tesque).

The study was conducted on 1000 whole adult mites and eggs, which were washed to reduce bacterial contamination, by modification of a previously reported method^{20 (link)}. Briefly, the raccoon dog’s skin and hair were washed at 37 °C for 1 h in washing solution [10% SDS, 0.1 μg/μL papain (Wako Pure Chemical Industries, Osaka, Japan), Pronase/ethylenediaminetetraacetic acid (EDTA) solution (Kyokuto Pharmaceutical Industrial, Tokyo, Japan)^{32 (link)} containing 25 μg/mL kanamycin (Nacalai Tesque, Kyoto, Japan) and 35 μg/mL chloramphenicol (Wako Pure Chemical Industries)]. Undissolved mites and eggs were then collected using a filter (EASYstrainer, 50 μm mesh, Greiner Bio-One, Kremsmünster, Austria).

The lung adenocarcinoma cell line (A549) was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation. Institute of Tropical Medicine, Nagasaki University supplied us with L. major, which was used as endorsed by the ethical board of the Institute of Tropical Medicine, Nagasaki University, Japan. M199 and Dulbecco’s Modified Eagle’s Media, kanamycin, dimethyl sulfoxide (DMSO), etoposide, miltefosine, fetal bovine serum, and MTT were obtained from Nacalai Tesque in Kyoto, Japan. The 96-well plates were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Solvents for the extraction, fractionation, and mass analysis were obtained from Sigma-Aldrich Inc., St. Louis, MO, USA.

Ampicillin, kanamycin, and chloramphenicol were purchased from Nacalai (Kyoto Japan). Neomycin, gentamicin, and apramycin were purchased from Sigma-Aldrich Co. (MO, USA). All other chemicals were of analytical grade. Fortimicin sulphate was kindly supplied from Kyowa Hakko Co. (Tokyo, Japan). Restriction enzymes were purchased from Takara Bio, Inc. (Shiga, Japan) and New England BioLabs (MA, USA).

Expression was assessed in LB containing 50 μg/mL of kanamycin (Nacalai, Japan). Induction was performed using 0.5 mM isopropyl β-D-thiogalactopyrosidase (IPTG) (Nacalai, Japan) after reaching an OD₆₀₀ of ~0.8 and inducing for 16 h at 20 °C. The overexpression of the G. antarctica HSP70 was analyzed using 15% SDS-PAGE (Figure S4). The protein bands were visualized by Coomassie brilliant blue R250 staining (Nacalai, Japan). Purification was performed using prepacked Ni-NTA columns and AKTA Avant based on the manufacturer’s instructions (GE Healthcare, Marlborough, MA, USA). The purified GaHSP70-1 and GaHSP70-2 were analyzed for purity using SDS-PAGE (Figure S5).