Apoa1

Pellet samples were resuspended in an appropriate volume of Protein Loading Buffer (Cat Num. 00193861, Lonza BV), equal sample volumes were separated by SDS-PAGE on precast gels (Cat Num. 4561095, Bio-Rad US). In Fig. 5C, fifteen micrograms of purified exosomes ExoRef™ (Exosomis SpA) were used as control sample. Proteins were transferred onto a nitrocellulose membrane (Cat Num. 15249794, GE Healthcare). Western blotting was run with primary antibodies against Alix (Cat Num. sc-271975, Santa Cruz), Tsg101 (Cat. Num. ab30871, Abcam), CD9 (Cat Num. 555370, BD Pharmigen), GAPDH (Cat Num. 25778, Santa Cruz), APO-A1 (Cat Num. ab17278, Abcam) and HRP-conjugated mouse or rabbit secondary antibody (Cat Num. P0260, Cat. Num. P0448; Dako). Mild stripping protocol from Abcam (Abcam https://www.abcam.com/ps/pdf/protocols/stripping%20for%20reprobing.pdf) was applied to strip to re-probe the same membrane with different antibodies. Chemiluminescence was produced using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Cat Num. 34095, Thermo Fisher Scientific).

For western blotting, HepG2 cells were harvested in lysis buffer (10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 10% Glycerol) containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, USA). After sonication and centrifugation, the protein concentration was determined by the Bradford reagent assay. Finally, SDS sample buffer was mixed with lysate. Secretome samples were dissolved in lysis buffer after lyophilization to the same concentration and mixed with SDS sample buffer. The samples were separated on a 6–16% gradient SDS-PAGE gel and transferred to a nitrocellulose membrane using the Hoefer wet transfer system. After blocking with TBS-T containing 5% skim milk for 30 min, immunoblotting was performed with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody for 1 h. The membranes were washed with TBS-T buffer and developed using ECL solution. Western blotting was performed in more than four biological replicates (n ≥ 4) with the antibodies against PDIA3 (Abcam, Cambridge, UK), APOA1 (Abcam, Cambridge, UK), THBS1 (Thermo Fisher Scientific, MA, USA), MIF (Thermo Fisher Scientific, MA, USA), FASN (Abcam, Cambridge, UK), and EEF2 (Abcam, Cambridge, UK).

Protein-content EV characterization was performed in accordance with the MISEV guidelines [17 (link)] using Western blotting, choosing markers from the different groups. To extract total proteins, EVs were lysed using M-PER mammalian protein extraction reagent (ThermoFisher) added with a 10 µL/mL protease inhibitor cocktail kit (ThermoFisher). Extracts were kept ice-cold and cleared by centrifugation at 20,000× g for 15 min at 4 °C. Protein concentration was determined using the Bradford method. A total of 30 µg of proteins were loaded on pre-cast gels (4–15% Mini-PROTEAN^® TGX™ Precast Gel, Biorad, Irvine, CA, USA), separated by electrophoresis, and transferred to nitrocellulose membranes. Specific antibodies to human CD63, CD81, ALIX, ApoA1, and GM130 (Abcam) were used for the immunoblotting of membranes. For specifications of the antibodies and dilutions see Supplementary Materials, Figure S1. The enhanced chemiluminescence (ECL) Western blotting protocol (Merck–Millipore, Burlington, MA, USA) was used to visualize the bands. Each characterization was repeated 3 times.

Tissues from pig cochleae were lysed with RIPA. Western blotting was performed similarly to previous studies [26 (link)]. Briefly, proteins separated by SDS-PAGE were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were treated with anti-NF-κB p65 (6956, Cell Signaling Technology, Inc) and Apo A1 (Abcam, ab64308) antibodies. Each antibody preparation was diluted in 5% skim milk, and protein bands were visualized using an ECL plus chemiluminescence detection system (WBKLS0500, Millipore, USA) and photographed.

Intestinal and liver tissues were prepared for Western blotting as described previously (25 (link)).
Proteins were separated on 8% SDS-PAGE gel and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked using defatted milk proteins, followed by the addition of primary antibodies directed against acetyl CoA carboxylase (ACC), carnitine palmitoyl transferase a (CPT1a), fatty acid synthase, AMP-activated protein kinase α (AMPKα) and phoshpoAMPK^Thr172 (pAMPKα) (1/1,000, Cell signaling), GAPDH, ABCA1, peroxisome proliferator activated receptor coactivator 1α (PGC-1α) (1/1,000, Abcam), Apo A1 (1/5,000, Abcam), microsomal triglyceride transport protein (MTTP), acyl-CoA dehydrogenase long chain (ACADL) (1/1,000, Thermo Fisher Scientific), PPAR-α (1/1,000, Cayman Chemical), phospho acetyl CoA carboxylase^Ser79 (pACC) (1/1,000, Millipore), Apo B (1/1,000, Sigma-Aldrich), and β-actin (1/250,000, Sigma-Aldrich). Bands were captured with the Chemidoc Imaging System and analyzed with the ImageLab software (Bio-Rad). For every protein of interest, β-actin or GADPH was used for sample normalization.