Iblot semi dry blotting system

Western blots were performed on protein extracts collected in Laemlli Buffer (Sigma) supplemented with an additional 10% SDS to comply with local controls for the removal of samples from containment level 4. Samples were heated at 95 °C for 10–15 minutes prior to removal to containment level 2. Protein lysates were loaded on to Novex 4–12% gradient acrylamide gels (Life Technologies). The gels were run in a Novex X-2 vertical gel tank for 50 minutes at 200 V in MOPS buffer (Life Technologies). PVDF blots were performed using the IBlot semi-dry blotting system (Life Technologies) and blots blocked for minimum 2 h at room temperature with non-fat dried milk (NFDM; Sigma) in Tris-Buffered Saline–Tween20 (TBST; Sigma). Blots were rinsed in TBST and primary antibody staining performed overnight with the IRF-3 and GAPDH specific rabbit polyclonal antibodies (Millipore). Secondary antibody staining was performed as per antibody instructions using goat anti-rabbit antibody conjugated to HRP. Luminescence assay was performed with ECL Prime reagent (GE Healthcare) after a 5 minute incubation and imaged in a GeneSys Gel Imaging System (GeneSys).

The N. benthamiana leaf samples were harvested 48 h after infiltration. Total protein was extracted from powdered plant tissue using a phenol solution (0.5 M Tris pH 9.4, 50 mM EDTA, 0.7 M sucrose, 0.1 M KCl containing 2% ß-mercaptoethanol and complete protease inhibitors (Roche)). The protein concentration was measured using a Bradford assay following the manufacturer’s instructions (Bio-Rad). 0.1 mg protein of each sample was separated on Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membranes using an iBlot (semi-) dry blotting system from Life Technologies. The blotting membrane was blocked with 5% BSA (bovine serum albumin) in TBST solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The membrane was incubated with 1:3000 diluted anti-GFP primary antibody (Sigma) for 3 h at room temperature. The membrane was washed with TBST solution and incubated with a 1:3000 dilution of anti-mouse IgG antibody (Sigma). The signal was detected using an AP conjugate substrate kit (Bio-Rad).

The N. benthamiana leaf samples were harvested 4 days after infiltration. Total protein was extracted from plant tissue powder using phenol solution (0.5 M Tris pH 9.4, 50 mM EDTA, 0.7 M sucrose, 0.1 M KCl containing 2% Mercaptoethanol and protease inhibitors cOmplete (Roche)). The protein concentration was measured using a Bradford assay following the manufacturer’s instructions (Bio-rad). Equal amounts of protein samples were separated on Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membranes using an iBlot (semi-) dry blotting system from Life Technologies. The blotting membrane was blocked with 5% BSA (bovine serum albumin) in TBST solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween20) for 1 hour at room temperature. The membrane was incubated with a 1:5000 dilution anti-HA primary antibody (Sigma) for 3 hours at room temperature. The membrane was washed with TBST solution and incubated with a 1:30000 dilution of anti-mouse IgG (Fc specific)-alkaline phosphatase antibody (Sigma). The signal was detected using an AP conjugate substrate kit (Bio-rad). Western blots were replicated using protein extracts from three independent isolations.