Nbt bcip buffer

Total protein from plant samples was extracted and separated by 12% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), as reported previously. After transfer onto nitrocellulose (Amersham, Uppsala, Sweden) by wet electroblotting, the proteins were detected with primary antibody to GFP or PVX and secondary anti‐mouse antibody (Sigma‐Aldrich, St Louis, USA). The antigen–antibody complexes were visualized using nitrotetrazolium blue chloride/ 5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT/BCIP) buffer (Sigma) under standard conditions.

Total proteins were extracted from plant samples using lysis buffer (100 mM Tris‐HCl, pH 8.8, 60% SDS, 2% β‐mercaptoethanol) and separated in a 12% SDS‐PAGE gel as previously described (Jiang et al., 2014), then transferred onto nitrocellulose (Amersham, Uppsala, Sweden) by electroblotting, and detected with primary antibody to GFP and HA‐tag and secondary anti‐mouse and anti‐rabbit antibodies (Sigma‐Aldrich, St Louis, MO, USA). The antigen‐antibody (for secondary anti‐mouse) complexes were visualized using NBT/BCIP buffer (Sigma) at room temperature and the antigen‐antibody (for secondary anti‐rabbit) was visualized using ECL HRP Chemiluminescent Substrate (Invitrogen) under a chemiluminescence analyzer.

A mixture of total proteins from at least three different samples was extracted with lysis buffer (100 mM Tris–HCl pH 8.8, 60% SDS, 2% β-mercaptoethanol). Briefly, 40 mg plant samples were lysed in 100 μl lysis buffer and placed on ice for 30 min. Protein samples were then centrifuged at 13,000 rpm for 15 min at 4°C, and then the supernatant was removed by aspiration and boiled. Seven microliters of protein was separated on 12% SDS-PAGE gels for detection with primary antibodies (anti-flag (0912-1, Huabio, China), anti-GFP (ET1607-31, Huabio, China), or anti-TuMV-CP (1075-06, Adgen, United Kingdom) and secondary antibodies (antimouse or antirabbit) (Sigma-Aldrich, St. Louis, MO, United States). Dilution rates were 1:2,500 for primary antibodies and 1:10,000 for secondary antibodies. After incubation with a secondary antibody, proteins were visualized with NBT/BCIP buffer (Sigma) at room temperature. The loading control was visualized by the band intensity of the internal reference protein Rubisco stained with a fuchsia dye. The relative amount of accumulated protein was calculated by comparing the protein band intensity with the loading control using Image J.

Ovaries were quickly dissected from Ae. albopictus adult females at 7, 14, and 21 days postinjection and then homogenized in radioimmunoprecipitation assay lysis buffer. Protein concentrations were measured using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. After the addition of 6× SDS loading buffer, the lysates were boiled for 10 min. The proteins of Ae. albopictus ovaries were separated by electrophoresis on a 12% SDS-PAGE gel running at 80 to 130 V for 2 h and then transferred to a polyvinylidene difluoride membrane. After this, the membrane was probed with primary antibodies (1:5,000) and tested using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA). In the experiment, anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) polyclonal rabbit serum (1:10,000, Aksomics, Shanghai, China) was used to monitor equal protein loading. Nitrotetrazolium blue chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) buffer (Sigma-Aldrich) was used to visualize target fragments under room temperature conditions. Anti-ZIKV (SPH2015) envelope antibody (Novus Biological) and anti-Wolbachia hsp60 antibody (Sigma-Aldrich) were used in this study.