Bradford based protein assay

At the end of experimental protocol, cardiomyocytes were homogenized by sonication (twice for 5 sec.) on ice in 50 mM Tris‐HCl (pH 7.4), containing 3.1 mM sucrose, 1 mM DTT, 10 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 2 μg/ml aprotinin and 0.1% Triton X‐100. Homogenates were centrifuged at 10,000 × g, for 10 min., at 4°C, and the supernatant was stored at −80°C for further biochemical analysis.
The total protein content in cardiomyocyte extracts was analysed by the Bradford based protein assay from Bio‐Rad (Hercules, CA, USA) and standardized with bovine serum albumin.

Interleukin (IL)-6, IL-10, IL-12p70, interferon (IFN)-γ, monocyte-chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured by cytometric bead array (CBA) multiplex assay (BD Biosciences, San Jose, CA) in accordance with the manufacturers' recommendations. Thrombin-antithrombin complexes (TATc; Siemens Healthcare Diagnostics, Marburg, Germany) and D-dimer (Asserachrom D-dimer, Roche Woerden, the Netherlands) were measured with commercially available ELISA kits. Protein levels in BALF were measured using a Bradford-based protein assay (Bio-Rad Laboratories, Hercules, CA). Aspartate aminotranspherase (ASAT) and alanine aminotranspherase (ALAT) were determined with commercial available kits (Sigma-Aldrich, St. Louis, MO), using a Hitachi analyzer (Boehringer Mannheim, Mannheim, Germany) according to the manufacturers' instructions.

2 × 10⁵ MSC were seeded in 6-well plates and incubated for 48 h in 2 ml of a mixture of equal volumes of growth medium and CM or in 2 ml of growth medium supplemented or not with the indicated cytokines. After washing with PBS, MSC were induced to undergo differentiation by incubation in osteogenic medium up to 21 days. As controls, untreated cells were incubated in growth medium. Media were partially replaced every 3 days. After 14 days of culture, cell layers were extracted with 5 × 10⁻¹ M NaCl, 5 × 10⁻² M Tris-HCl pH 8.0, and 1% Triton X-100 and supplemented with a mixture of protease inhibitors. Alkaline phosphatase (ALP) activity was assessed in cell layers by determining the release of p-nitrophenol from p-nitrophenyl phosphate (Sigma). The data were normalized to the total protein contents determined by a Bradford-based protein assay (Bio-Rad Laboratories Inc., Hercules, CA). The degree of cell layer calcification was assessed in cells cultured for 3–21 days using alizarin red staining. Briefly, cells were fixed with ethanol and stained with 40 mM alizarin red S in deionized water at pH 4.2. Images of stained cell layers were obtained using a phase-contrast microscope. The bound stain was eluted with 10% (w/v) cetylpyridinium chloride and the absorbance at 562 nm was measured using a spectrofluorometer.

Total protein extracts from yeast were prepared as previously described (13 (link)). Protein concentration was determined with a Bradford-based protein assay (Bio-Rad) using bovine serum albumin as standard. A total of 20 μg protein was loaded per lane for SDS–polyacrylamide gel electrophoresis (SDS-PAGE).
For total protein extracts from plants, leaves from 4-week-old rosettes were harvested and snap frozen in liquid nitrogen. Glass homogenizers were used to homogenize the plant material in extraction medium [1 ml/100 mg of fresh weight; 40 mM tris-HCl (pH 6.8), 5 mM MgCl₂, 2% (w/v) SDS, and complete protease inhibitor (Roche)]. Insoluble debris was pelleted by centrifugation (5 min, 4°C, 20,000g) before loading equal sample volumes for SDS-PAGE.
For immunoblotting, proteins were transferred onto low-fluorescence polyvinylidene difluoride membranes following SDS-PAGE and probed with antibodies specifically recognizing ESV1, LESV, or green fluorescent protein (GFP). Antisera against Arabidopsis ESV1 and LESV (14 (link)) were used at concentrations of 1:200 for affinity purified anti-ESV1 and 1:3000 for anti-LESV crude serum. YFP-tagged proteins and plant actin (used as a loading control) were detected using commercial antibodies (anti-GFP: Torrey Pines Biolabs, TP401, at a concentration of 1:5000; anti-actin: Sigma-Aldrich, clone 10-B3, at a concentration of 1:10,000).

Frozen AT (~100 mg) was ground in a mortar with liquid nitrogen. Per microgram of grounded powder, 2 μl of 50 mM ammonium bicarbonate with 5 M urea was added to dissolve the powder. The solution was freeze-thawed in liquid nitrogen 3 times after which it was vortexed for 5 min. The homogenate was centrifuged at 20,000 g for 30 min at 10 °C. The supernatant was carefully collected and protein concentrations were determined with a Bradford-based protein assay (Bio-Rad, Veenendaal, the Netherlands). A control sample was prepared from a pool of 10 μl of each sample.
Samples were digested with Trypsin (Promega) and peptides from 100 µg protein were labelled with TMT isobaric mass tagging labelling reagent (10-plex; Thermo Scientific, West Palm Beach, FL, USA) according to the manufacturer’s protocol. Briefly, 100 μg of protein was used for each sample. The TMT labeling reagents were dissolved in 41 μl acetonitrile per vial. The reduced and alkylated samples and control were added to the TMT reagent vials. The reaction was incubated for 1 h at room temperature and quenched for 15 min by adding 8 μl of 5% hydroxylamine, as described previously^{19 (link)}. Equal amounts of the 16 samples from 8 subjects (biological replicates) were combined and analyzed by LC-MS in two injections; each injection composed of a mixture of 8 samples from 4 subjects and the control.