Ab20737

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).

Two microliters of plasma from 12 h-fasted mice fed a WTD for 9 weeks were diluted with RIPA buffer (1:5, v:v) under reducing and denaturing conditions, separated by 4.5% SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. The blot was incubated with rabbit polyclonal anti-ApoB antibody (1:1,000, ab20737, Abcam, Cambridge, UK) and HRP-conjugated goat anti-rabbit (1:5,000) (Dako, Glostrup, Denmark) was visualized by enhanced chemiluminescence detection (Clarity Western ECL substrate; Bio-Rad) on a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA) and quan-tified using ImageJ software.

Serial sections (5 μm thick) of paraffin‐embedded mouse aortas were placed on poly‐L‐lysine‐coated slides, deparaffinized and treated with H₂O₂ for suppression of endogenous peroxidase activity. Immunohistochemical analysis was performed with the avidin‐biotin immunoperoxidase technique, as previously described 56. Samples were analysed for Apo B (rabbit‐polyclonal anti‐apolipoprotein B; dilution 1:100; Abcam, ab20737, Abcam: Cambridge, UK), LRP5 (rabbit‐polyclonal anti‐LRP5; dilution 1:50; Abcam, ab38311) and MAC387 that is a marker for tissue infiltrating monocytes 57, 58 [anti‐Macrophage antibody (MAC387); dilution 1:100; Abcam, ab22506]. Images were captured and digitized using a Nicon Eclipse 80i microscope and a Retiga 1330i Fast camera. Controls without primary antibodies were run with each set of specimens and showed no labelling background.

The total protein of tissues and cells was extracted by high-efficiency RIPA lysis buffer (R0010, Solarbio). After centrifugation at 15,000 rpm/min for 15 min and lysis at 4°C for 15 min, the supernatant was harvested, and the protein concentration was evaluated employing the bicinchoninic acid kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Following separation utilizing polyacrylamide gel electrophoresis, the protein was transferred onto PVDF membranes which were sealed at 5% BSA for 1 h at ambient temperature and probed with the diluted primary antirabbit antibodies (all from Abcam) against Sesn2 (ab178518, 1: 10000), Srx1 (ab203613, 1: 10000), Trx1 (ab273877, 1: 10000), Beclin-1 (ab210498, 1: 10000), LC3A/B (ab128025, 1: 1000), Ki67 (ab92742, 1: 10000), Aggrecan (ab3778, 1: 10000), MMP3 (ab39012, 1: 10000), GRP94 (ab238126, 1: 10000), APOB (ab20737, 1: 10000), and GAPDH (ab8245, 1: 10000) overnight at 4°C. The next day, the membrane was reprobed with HRP-labeled goat antirabbit IgG (ab205718, 1: 20000, Abcam) for 1 h at ambient temperature. The developing solution was added for development. ImageJ 1.48 software (National Institutes of Health) was employed for protein quantitative analysis, and protein quantitative analysis was implemented with the gray value ratio of each protein to the internal reference GAPDH.

To quantify aldosterone and C-reactive protein (CRP) in plasma, an aldosterone ELISA kit (ab136933) and rat CRP ELISA kit (PTX1, ab108827), respectively, were obtained from Abcam (Cambridge, UK). Appropriately diluted plasma samples were reacted using the kit in according to the manufacturer’s suggestion.
The composition of apolipoprotein/lipoprotein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an exact amount of protein loaded in the gel (5 µg of total protein per lane) from individual HDL₃, and the levels of apolipoprotein expression were measured by immunodetection. Anti-human apoA-I antibody (ab7613) and anti-apo-B antibody (ab20737) were ordered from Abcam (Cambridge, UK). Relative band intensity (BI) was compared through band scanning with Chemi-Doc^® XRS+ (Bio-Rad, Hercules, CA, USA) using Image Lab software (Version 5.2, Bio-Rad, Hercules, CA, USA). Blot images are imported into the Quantity One software and then contrast was adjusted in a way so that the bands were fully noticeable on the blot image. The area around the band was chosen; additionally, the background intensity was deducted from the blot image. The appropriate bands were used to calculate band intensities and exported to excel for analysis.