Nc filter membrane

The recombinant protein VP4/VP56/VP35 was analyzed on 10% SDS-PAGE and by WB. The protein samples were separated by 10% SDS-PAGE and the gels were stained using Coomassie Brilliant Blue G-250 (C.I.42655, Sigma, Shanghai, China). For the WB, the gels were transferred onto a nitrocellulose (NC) filter membrane (Millipore, Burlington, MA, USA). The membrane was then blocked with 5% skim milk in phosphate-buffered solution-Tween-20 (PBST, Boster, Wuhan, China) for 2 h and washed with PBST. Afterward, the mouse anti-His-tag/anti-GST-tag monoclonal antibody (1: 3000, ABclonal, Wuhan, China, AE003/AE001) was added and the incubation continued for 2 h. The membrane was then rewashed with PBST and incubated with a 1: 2000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (ABclonal, Wuhan, China, AS064) for 1 h. Following another wash with TBST, the NC filter membrane was stained with Clarity TM Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and y imaged using the Amersham Imager 600 (Little Chalfont). Finally, the purified proteins and a control sample (10 μL culture supernatant of GS115 transfected with p9K) were also detected using the same method.

Genomic DNA of L. incisa was double digested with XhoI/NotI or HindIII/NotI restriction endonucleases at 37°C for 4-6 h. The digested DNA samples were fractionated on a 1.0% agarose gel and then transferred to a NC filter membrane (Millipore). A pair of primers was designed based on the conserved domain of GPAT (Supplementary Table S1). A 311-bp biotin-labeled DNA sequence was prepared to use as a probe with a North2South^® Biotin Random Prime Labeling Kit (Thermo Scientific). Subsequently, the hybridization was detected by the standard Southern blot procedure (Sambrook and Russell, 2001 ) with a North2South Chemiluminescent Hybridization and Detection Kit (Thermo Scientific). Signals were visualized by exposure to XBT-1 film (Kodak) at room temperature for 60-120 s.