Imagequantlas 4000 digital imager

Cells and tissues were collected and lysed in 40 μl RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Protein concentration was determined with the BCA Protein Assay (ThermoFisher). Protein extracts (30–40 μg) were mixed with Novex Sample Buffer and Reducing Agent (Life Technologies), resolved by SDS-PAGE in 12%, 10% or 4–12% gradient gels and blotted onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked for 1 h with 5% non-fat dry milk (Bio-Rad) or 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and then incubated overnight at 4 °C with the following primary antibodies: mouse anti-β-actin (1:3000; Sigma-Aldrich); mouse anti-FABP4 (1:500, Santa Cruz Biotechnology); rabbit anti-adiponectin (1:1000; Thermo-Fisher Scientific); rabbit anti-Emilin-2 (1:1000 [45 (link)], antibody specificity shown in Additional file 1: Fig. S2F). After three washes for 10 min in TBS-T, membranes were incubated for 1 h with goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:1000; Bethyl). After three further washes for 10 min in TBS-T, bands were detected with LiteAblot Extend chemiluminescent substrate (Euroclone), using a ImageQuantLAS 4000 digital imager (GE Healthcare).

Cells and EVs were lysed in 1% SDS and 10 mM EDTA lysis buffer at 100°C for 5 min. Samples were subsequently boiled at 100°C for 5 min in 1× NuPAGE LDS sample buffer (Novex NP0007) with 10% 1M DTT (Promega 0000085706), and 20 μg of protein per sample was loaded onto a 4–15% Mini‐PROTEAN TGX gel (Bio‐Rad 4561083). Proteins were transferred to a PVDF membrane using wet transfer, and the membrane was blocked using 5% milk. Membranes were probed for calnexin (Abcam ab22595), cytochrome‐C (BD Biosciences 556433), and GM130 (BD Biosciences 610822), incubated with anti‐mouse HRP (GE Healthcare NA931V) or anti‐rabbit HRP (GE Healthcare NA934V), and proteins were detected using ECL Western Blotting Substrate (Pierce 32209) in combination with an ImageQuant LAS 4000 digital imager (GE Healthcare). As a reliable loading control for cells and EVs has not yet been identified, equal protein loading was confirmed using Ponceau S staining and lanes were quantified using ImageJ.