Immobilon p membrane

Non-infected, wild type (WT) TMV and MBP TMV infected N. benthamiana leaves were harvested for western blotting 16 days after inoculation. Approximately 30 mg of leaf material was ground in liquid nitrogen, combined with 100 μl of 2X Laemmli loading buffer and then boiled for 5 min to denature the proteins. After centrifugation at 16,000 rpm, 20 μl was loaded onto 15% SDS-PAGE gels, along with 15 μl of Novex prestained ladder (Invitrogen, Paisley, UK). Gels were electroblotted onto Immobilon-P membrane (Millipore, Watford, UK). The Immobilon-P membrane was blocked by incubating in 1X PBS, 1% BSA and 0.05% Tween, for 1 h. Anti-TMV antibodies raised in rabbits were added to the membranes in blocking solution at a 1/10000 dilution and incubated with shaking at room temperature for 1 h. After washing, an anti-rabbit IgG alkaline phosphatase conjugate was added to the blots at a concentration of 1/1000 in blocking buffer (A8025-1ML; Sigma, Dorset, UK). After incubation at room temperature under shaking conditions the blots were washed and covered with BCIP/NBT (B1911; Sigma, Dorset, UK). The blots were left to develop for 10 min, after which banding was visible. The developed blots were scanned and saved as jpeg images.

Cell homogenates were prepared as described by Gong et al. [30 (link)]. Immunoblots were performed essentially as described in our previous studies [31 (link)]. The protein concentrations of homogenates were determined using the method of Bradford [32 (link)] (Bio-Rad, Richmond, CA, USA). Aliquots of cell homogenates containing 100 ng–50 µg of protein or 1–30 µL of extracellular vesicles were separated by SDS-PAGE on gels containing 8% acrylamide (for VE-cadherin), 14% (for CD63 and CD81) or 10% (for all other protein targets). Polyacrylamide gels were run under reducing conditions or non-reducing conditions (for CD63, CD81 and VE-cadherin). Proteins were blotted onto Immobilon-P membranes (SIGMA-Aldrich). ProSieve Protein Colored Markers standards (Lonza Walkersville, Inc.) were used to calibrate the gels. For most targets, immunoblots were developed using enhanced chemiluminescence reagents (ECL Prime, GE Healthcare Biosciences, Pittsburgh, PA, USA); Alix and CD81 blots were reacted with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc.). Chemiluminescence was detected by exposing blots to X-ray film.

The following antibodies were used: monoclonal mouse anti-human fibronectin antibody number 42042, purchased from QED Bioscience Inc., San Diego, California, USA, and goat anti-rabbit immunoglobulin G number A5420, conjugated with horseradish peroxidase, obtained from Sigma-Aldrich, Germany. The following reagents were applied: sodium metaperiodate and hydrazide LC-biotin obtained from Thermo Scientific, USA; standard fibronectin from human plasma, DMSO (dimethyl sulfoxide), sodium dodecyl sulfate, Triton X-100, Brilliant blue R250, glycine, Immobilon P membranes, dithiothreitol, Tween 20 (polyoxyethylene sorbitan monolaurate), and TMB (3.3′,5,5′-tetramethylbenzidine), all supplied by Sigma-Aldrich, Germany; Sephadex G-25 obtained from Pharmacia, Sweden; HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) supplied by Fluka, Germany; BLOT-QuickBlocker purchased from Millipore, USA; TEMED purchased from ICN Biomedicals, USA; streptavidin-coated sensor chip (SAP) obtained from XanTec Bioanalytics, Germany. All of the remaining, applied reagents were supplied by POCh Gliwice, Poland.

Cells were washed with ice-cold PBS and lysed in sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT, and 0.004% bromophenol blue). Cell lysates were then subjected to SDS-PAGE on 4%–20% (Bio-Rad Norway AS. Oslo, Norway) gradient gels and blotted onto Immobilon-P membranes (Sigma-Aldrich, Oslo Norway). Membranes were incubated with goat anti-human STEAP1 antibody (sc-10262; Santa Cruz Biotechnology, Heidelberg, Germany) or mouse anti-human vinculin monoclonal antibody (Sigma-Aldrich, Oslo, Norway) at 4°C overnight. Next, the membranes were washed and incubated with the fluorescently labeled secondary antibodies IRDye 680RD donkey anti-goat immunoglobulin G (IgG) and IRDye 680RD donkey anti-mouse IgG (LI-COR, Homburg, Germany) and analyzed by Odyssey infrared scanner (LI-COR, Bad Homburg, Germany).

Cells were lysed by suspension (1 h at 4°C) in the lysis buffer A containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 150 mM NaCl and protease and phosphatase inhibitor cocktails. Protein concentration was determined by Bradford method. Proteins (20-30 μg) were subjected to 9% or 11% SDS-PAGE, blotted on Immobilon-P membranes (Sigma-Aldrich), processed in western blot with the indicated antibodies and developed using an enhanced chemiluminescent detection system (ECL). Immunostained bands were quantified by means of a Kodak-Image-Station 4000MM-PRO and analysis with Carestream Molecular Imaging software (New-Haven, CT).