Horseradish peroxidase conjugated goat anti rabbit secondary antibody

CD109 expression was assessed using CD109 immunohistochemical staining. The slides were then dewaxed in xylene and dehydrated in ethanol. Staining was performed using BondMax-autostainer and other reagents (Leica Microsystems, Berlin, Germany). Deparaffinization was performed automatically in the autostainer with BondWash solution (Leica Microsystems) at 72°C for 30 minutes. After washing, the slides were incubated overnight at 4°C with a rabbit polyclonal anti-CD109 antibody (dilution 1:50, cat. No., HPA009292, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution 1:100, cat. No., SA00001-2, ProteinTech Group, Inc., Wuhan Sanying Biotechnology, Wuhan, China) for 90 minutes at room temperature. Antibody binding was performed by incubating the slides for 1 minute with a solution of 1 drop of 3,3'-diamino-benzidine (20 ×) per 1.0 mL diamino-benzidine substrate buffer (cat. No. ZLI-9017, Origene Technologies, Inc., Beijing, China). The slides were then counterstained with EnVision FLEX hematoxylin (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) for 1 minute and dehydrated using ethanol and xylene. For negative control, staining was performed without primary antibody.

Total protein was extracted from 1 g agro‐infiltrated N. benthamiana leaf tissue in 2 ml extraction buffer, as previously described (Zhu et al., 2017 (link)). After centrifugation at 18,000 ×g and 4°C for 30 min, 10 µl supernatant was mixed with 5 µl 3× SDS loading buffer and then subjected to 12.5% SDS‐PAGE. For YFP‐Sw‐5b detection, 1.5 ml supernatant was incubated with GFP Trap beads (Chromotek) at 4°C for 90 min. The beads were heated in 30 µl 1× SDS loading buffer at 95°C for 10 min, and protein samples were separated by 10% SDS‐PAGE. The proteins were transferred through electroblotting from the gel to a polyvinylidene fluoride membrane (GE Healthcare), which was blocked with 5% skim milk solution for 1 h and incubated with anti‐TSWV N (1:5000, produced in our laboratory), anti‐TSWV NSm (1:5000, produced in our laboratory), or anti‐YFP (1:5000, produced in our laboratory) primary antibodies for 1.5 h at room temperature. After incubation with horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (1:10,000, Sigma‐Aldrich) for 1 h, the blots were detected using the ECL Substrate Kit (Thermo Scientific) by the ChemiDoc Touch Imaging System (Bio‐Rad). To estimate protein loading, the blots were stained with Ponceau S.

Total protein of HepG2 cells was extracted according to the manufacturer's instructions using RIPA lysis buffer containing protease inhibitors (Promega Corporation). Protein concentration was determined using bicinchoninic acid assay kit (Bio-Rad Laboratories, Inc.). Proteins (30 µg per lane) were separated using 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. Following blocking with 5% skimmed milk in TBS at room temperature for 2 h, the membrane was incubated with rabbit anti-Axin2 polyclonal antibody (1:1,000; cat. no. ab32197; Abcam), rabbit anti-β-actin polyclonal antibody (1:5,000; cat. no. ab8227; Abcam) and rabbit anti-GAPDH polyclonal antibody (1:5,000; cat. no. ab37168; Abcam) for 12 h at 4°C. The membrane was washed three times in TBS + Tween-20 (0.1% V/V) and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; Sigma-Aldrich; Merck KGaA) at room temperature for 2 h bands were detected using enhanced chemiluminescence substrate, and relative expression of proteins was normalized to GAPDH using Scion Image v. 4.0.2 software (Scion Corporation).

Transfected cells were washed using PBS. Subsequently, total protein was extracted from the cells using ice-cold RIPA buffer. Following homogenization, centrifugation (3,000 × g, 4°C, 60 min) was performed and the supernatant was collected. Protein determination was performed via BCA assay. Total protein (50 µg) was separated via 12% SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skimmed milk (room temperature, 60 min). Subsequently, the membranes were incubated at 4°C for 24 h with the following primary antibodies: Anti-TOB1 (1:1,000; ProteinTech Group, Inc.) and an anti-GAPDH (1:2,000; ProteinTech Group, Inc.). Following primary incubation, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence substrates (EMD Millipore). Protein expression was quantified using Bio-Rad Gel Doc XR+ system (Bio-Rad Laboratories, Inc.).