Cdp star detection reagent

Total RNA was extracted from cells transfected with HBV plasmids using TRI Reagent. After treatment with DNase I and RNase inhibitor, RNA samples were separated on 1.2% agarose gel with 7% formaldehyde at 60 V for 3 h in 1 × 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (20 mM MOPS, 5 mM sodium acetate and 2 mM EDTA). The samples were transferred to a nylon membrane (Roche Diagnostics, Tokyo, Japan) with 20x SSC transfer buffer for 16 h, and subsequently cross-linked to the membrane by ultraviolet light (120 mJ/cm²). After washing, the blotted membrane was dried at room temperature. The blot was prehybridized with DIG Easy Hybridization buffer (Roche Diagnostics) in 68 °C and hybridized with an appropriate DIG-labeled RNA probe labeled with DIG-11-UTP at 68 °C overnight using the DIG Northern Starter Kit (Roche Diagnostics). To generate a DIG-labeled RNA probe with specific binding to HBV pregenome and HBs RNA, PCR fragments covering the nt 1998–2447 and nt 3205–488 regions were used as templates for in vitro transcription for the pregenome probe and HBs probe, respectively. RNA was labeled in the T7 promoter transcriptional system with DIG-11-UTP using a labeling mixture from the DIG Northern Starter Kit (Roche Diagnostics). Detection of the DIG-labeled probe on the blot was performed using CDP-Star detection reagent (GE Healthcare, Tokyo, Japan).

The isolation of genomic DNA from mosquitoes and the Southern blotting analysis were performed as described previously [49 ]. Genomic DNA was digested with Msp I, separated on a 0.8% agarose gel, and then transferred to a Hybond-N+ membrane (GE Healthcare UK Ltd., Buckinghamshire, UK). Probe labeling and the detection of signals were performed using AlkPhos Direct labeling Reagents and CDP-Star Detection Reagent (GE Healthcare UK Ltd.) according to the supplier’s protocol.

Genomic DNA was extracted from WT parasites and two tpx-1 KO clones. Ten micrograms of DNA was digested overnight with 100 units of HindIII, AatII, or BamHI and SalI. The DNA was separated by agarose gel electrophoresis and then transferred onto HyBond N+ membranes (GE Healthcare, Buckinghamshire, United Kingdom). Two probes were used, one corresponding to the complete gfp orf and the other to a 0.5-kb length of the cas9 orf, and both were labeled and hybridized by the use of an AlkPhos Direct kit (GE Healthcare). Chemiluminescent signal was developed with CDP-star detection reagent (GE Healthcare) and detected with a multipurpose charge-coupled-device (CCD) camera system (LAS-4000 mini EPUV; Fujifilm, Japan).

Total RNA from TcIL3000 BSF, PCF, EMF and MCF was extracted using RNA extraction reagent (Cat. No. 15596–018, Thermo Fisher Scientific, Hudson, USA) according to the manufacturer’s instructions. Ten micrograms of total parasite RNA was separated on a 0.8 % agarose gel containing 2.2 M formaldehyde in 3-[N-morpholino] propanesulfonic acid (MOPS) buffer. The RNA was transferred onto a nylon membrane (GE Healthcare Bio-Sciences Corp.) and then fixed to the membrane by UV-induced crosslinking. The transferred RNA was probed with alkaline phosphatase-labelled DNA probes (GE Healthcare Bio-Sciences Corp.) under high-stringency conditions. The DNA probes to detect TcHpHbR mRNA and the reference transcript (18S ribosomal RNA) were prepared by a PCR using the primers shown in Table 1 [17 (link)]. Probe binding was visualized with CDP-STAR detection reagent (GE Healthcare Bio-Sciences Corp.) according to the manufacturer’s instructions.

The total protein was isolated from vegetative hyphae using a previously described method [23] (link). The protein separated on SDS–PAGE gels was transferred onto a PVDF membrane using an Xcell SureLock Mini-Cell. The phosphorylation activation of Maf1 and Cmk1 MAPK kinase was detected using a PhosphoPlus p44/42 MAP kinase antibody kit (Cell Signaling Technology). Alkaline Phosphatase-conjugated secondary antibody and light emission from the enzymatic dephosphorylation of the CDP-Star Detection Reagent (GE Healthcare, Tokyo, Japan) was detected using the Fujifilm LAS-1000 Plus Gel Documentation System (Fujifilm, Tokyo). Anti-actin antibodies (Wako, Japan) were used at a 1∶1000 dilution for Western blot analysis.