Anti gfp

Equal volumes of lysate were loaded for each sample onto 4–12% Criterion XT Bis-Tris protein gels in 1x XT MES buffer. Protein was transferred to PVDF membrane via turbo blot. Membranes were then placed in 2.5% milk, 1x TBST blocking solution for 1 hr. Primary antibody was used in 1x TBST at either 1:50,000 for anti-eEF2 (Kerafast, rabbit), or 1:5000 for anti-PGK1 (Invitrogen, mouse), anti-HA (Roche, rat), anti-FLAG (Sigma, mouse), and anti-GFP (Takara, mouse) and incubated on a rotator overnight at 4°C. Membranes were washed in 1x TBST three times, 10 min each. The corresponding HRP-conjugated secondary antibody was added to the membrane at 1:5000 in 1x TBST and incubated for 1–2 hr. Membranes were washed three times for 10 min each. Pico solution (details) was added to membranes for approximately 3 min and then membranes were scanned using a G:BOX Chemi XX6 (Syngene) with varying exposure times.

Whole cell lysates were prepared from monolayers of infected CESCs, non-infected CESCs or VP22 expressing LMH by resuspending the cells in laemmli 2× sample buffer. Solubilized proteins were separated on a 10% SDS-PAGE. Resolved proteins were transferred to nitrocellulose membrane, blocked in Tris 10 mM pH 8.25, NaCl 150 mM, Tween 0.2% containing milk 3% and subsequently incubated with the mouse monoclonal anti-MDV VP5 (F19), the rabbit polyclonal serum anti-PRV VP22, the mouse monoclonal anti-MDV VP22 (L13a), the rabbit polyclonal anti-ILTV VP22, a rabbit polyclonal anti-GFP (# 632593, Takara, Shiga, Japan) or a mouse monoclonal anti-GAPDH (MAB374, Millipore, Bedford, MA, USA) primary antibody. Subsequently, goat anti-mouse IgG (#A3562, Sigma) and anti-rabbit IgG alkaline phosphatase-conjugated antibodies (#A3687, Sigma) or goat anti-mouse IgG (#A4416, Sigma) and anti-rabbit IgG (#A0545, Sigma) peroxidase-conjugated antibodies were used. Alkaline phosphatase and peroxidase signals were revealed using NBT-BCIP (Zymed, South San Franscisco, Calif, USA) or Immobilon Western Chemiluminescent HRP substrate (P90720, Millipore), respectively.

The following monoclonal and polyclonal antibodies or nanobodies were used in this study: anti-E-cadherin (C-terminus: BD Transduction Laboratories, 61018; N-terminus: Genetex/Biozol GTX134997), anti-Tsg101 (Abcam, 4A10), anti-GFP (Takara, 632592), anti-GFP-nanobodies (Chromotek), anti-actin (BD Transduction Laboratories, 612656), anti-Hrs (Enzo, A-5; GeneTex, GTX89364), anti-GAPDH (Abcam, 6C5), anti-giantin (Covance, PRB-114C), anti-PDI (BD Transduction Laboratories, 610946), anti-TOM20 (Santa Cruz, Sc11415).

The fresh hairy root samples were homogenized in 500 μL of plant protein extraction reagent containing 1X protease inhibitor cocktail, according to the manufacturer’s recommendation (CoWin Biosciences, China). The whole protein extracts from the H. niger transgenic lines and control lines were mixed with 5X sample loading buffer (50% glycerol, 10% SDS, 5% β-mercaptoethanol, 0.5% bromophenol blue, and 0.25 M Tris, pH 6.8), heated to 100 °C for 5 min, loaded onto a 10% SDS-polyacrylamide gel, and electrophoresed at 80 V for 120 min in a Bio-Rad Mini Trans-Blot cell. The total protein was subsequently transferred to PVDF membrane. The membranes were blocked using 5% non-fat dry milk in PBS-0.05% Tween-20 and incubated overnight with the primary antibody (anti-β-actin, Sigma, USA or anti-GFP, Takara, Japan) at a 1:5000 dilution in 3% Bovine serum albumin. The blots were washed three times with TBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.3% Tween-20 (V/V)) and incubated for 4 h with goat anti-mouse IgG antibody (1:5000) (Abmart, China). The bands were developed using a chemiluminescent substrate (Thermo, USA).