Ab227455

Immunodepletion of lipoprotein particles from the EV fractions was performed using antibodies against lipoproteins and Protein A-conjugated magnetic beads (Dynabeads, Invitrogen). Protein A beads were washed with 0.001% Tween 20 containing PBS supplemented with 0.1% bovine serum albumin (BSA), and incubated overnight with antibodies against Apolipoprotein A1 (ApoA1) (ab227455, rabbit polyclonal, Abcam), and Apolipoprotein B (ApoB) (ab20737, rabbit polyclonal, Abcam) at 4°C. The beads were then incubated overnight with the EV fractions at 4°C. After the incubation, the supernatants and the beads were separated by DynaMag-2 magnet, and analyzed by Western blotting and nanoparticle tracking analysis.

Equal amounts of total proteins of the EV fractions were separated using 5% to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (ATTO), and transferred to PVDF membranes (Bio-Rad). The membranes were incubated overnight with the following primary antibodies at 4°C: anti-CD63 (10628D, mouse monoclonal, Thermo Fisher Scientific), anti-CD9 (CBL162, mouse monoclonal, Merck Millipore), anti-Hsc70 (ADI-SPA-819, rabbit polyclonal, Enzo Life Sciences), anti-GPIIb (LS-B13882, rabbit polyclonal, LifeSpan Biosciences), anti-GPIIIa (R30709, rabbit polyclonal, NSJ Bioreagents), anti-ApoB (ab20737, rabbit polyclonal, Abcam), and anti-ApoA1 (ab227455, rabbit polyclonal, Abcam) for both mouse and human samples, anti-PF4 (MAB595, rat monoclonal, R&D systems) for mouse samples, and anti-PF4 (ab129183, rabbit monoclonal, Abcam) for human samples. As secondary antibodies, horseradish peroxidase (HRP)-conjugated immunoglobulins were used. The HRP signal was visualized by ImmunoStar Zeta/LD (Wako), and captured using an Amersham Imager 600 system (Cytiva). Acquired images were analyzed using ImageJ software (NIH).

Western blot was carried out to validate the TMT-based proteomics analysis results. Skeletal muscle total protein was extracted with RIPA lysis buffer containing protease and phosphatase inhibitors, and the concentrations were determined using a BCA protein assay kit (P0010S, Beyotime, China). The total protein (10 μg) from each sample was separated by SDS-PAGE gel electrophoresis and then electrotransferred onto PVDF membranes. After 1.5 h of blocking in the blocking solution, the PVDF membrane containing the proteins was incubated in the desired primary antibody, including Anti-ApoA1 antibody (1:1000, ab227455, Abcam, UK), Anti-ApoA4 antibody (1:10,000, 5700S, Cell Signaling Technology, USA) and Anti-alpha Tubulin antibody (1:5000, ab7291, Abcam, UK) overnight at 4 ℃. After incubating with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:5000, SA00001-1, Proteintech, USA) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000, SA00001-2, Proteintech, USA) for 1 h, PVDF membrane was washed with TBST, incubated with an electro-chemiluminescence (ECL) developing solution, and exposed and photographed. Using ImageJ, target protein bands are measured and their relative levels of expression are quantified by the ratio of the corresponding protein to α-tubulin.