Liteablot kit

Plant tissue was ground in a mortar in liquid nitrogen or homogenized in a TissueLyzer bead mill (Qiagen). Total proteins were extracted in 150 mM Tris-HCl pH 7.5, 6 M urea, 2% (w/v) SDS, 5% (v/v) glycerol and 5% (v/v) β-mercaptoethanol; heat denatured (96°C, 5 min) and centrifuged (14000 rpm, 10 min) to remove cell debris. Protein samples were separated by glycine-SDS-PAGE and electroblotted onto a nitrocellulose membrane. Ponceau red staining was used to control protein loading equivalence. Proteins were detected using anti-PPV CP and anti-PPV HCPro rabbit sera, and anti-GFP monoclonal antibody (clones 7.1 and 13.1, Roche) as primary antibodies. Antibodies raised against tobacco class II PR-2 [44] (link) and class II PR-3 proteins [47] (link) were kindly provided by Dr. T. Heitz (IBMP-CNRS, Strasbourg, France). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson) or sheep anti-mouse IgG (GE Healthcare) were used as secondary antibody. Immunostained proteins were visualized by enhanced chemiluminescence detection with a LiteABlot kit (Euroclone). For signal quantification, chemioluminescence was acquired in a ChemiDoc XRS imager (BioRad) and analyzed with ImageJ software [110] (link).

For GP and VSV-M quantitation, PV preparations were cleared by brief centrifugation (1500 rpm, 5 min, 4°C) and purified by ultracentrifugation in 30% sucrose cushion (in PBS). Pellets were re-suspended in DMEM, and a fraction was lysed with Laemli buffer. Then, proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in 5% (wt/vol) skim milk in PBS, 0.1% (vol/vol) Tween-20 (PBST), membranes were incubated with primary antibody (Anti VSV-M monoclonal antibody from Merck, reference MABF2347, and anti HA high affinity antibody from Merck, reference 11867423001) in 5% (wt/vol) skim milk, PBST, for 2h at room temperature. Then, membranes were washed 3 times with PBST, and HRP-coupled secondary antibodies were applied 1:5000 in PBST (1 h, room temperature). Membranes were then washed with PBST, blots were developed by enhanced chemiluminescence (ECL) using LiteABlot kit (EuroClone). Signals were acquired by ImageQuant LAS 4000Mini (GE Healthcare Lifesciences). Signals were acquired with an ImageQuant LAS 4000Mini (GE Healthcare Lifesciences, Glattbrugg, Switzerland) instrument and quantified with ImageJ Software.

Proteins were separated by SDS-PAGE and transferred to nitrocellulose. After blocking in 5% (w/v) skim milk in PBS, 0.1% (v/v) Tween-20 (PBST), nitrocellulose membranes were incubated with primary antibody (1–10 µg/mL) in 5% (w/v) skim milk, PBST, in cold, for 12–16 h. Then, membranes were washed 3 times with PBST, and HRP-coupled secondary antibodies were applied 1:5000 in PBST (1 h, room temperature). After washing in PBST, blots were developed by enhanced chemiluminescence (ECL) using a LiteABlot kit (EuroClone, Pero, Italy). Signals were acquired by ImageQuant LAS 4000Mini (GE Healthcare Lifesciences, Glattbrugg, Switzerland).