Calnexin

Exosomes isolated from serum samples were resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitor cocktail (Millipore Corporation, MA) at 4 °C for 30 min. After sonication on ice, debris was removed by centrifugation at 12,000×g for 10 min at 4 °C. Protein concentrations were determined by BCA protein assay kit (Thermo Scientific, IL). Exosome extracts were separated on 4–20% Bis-Tris Nu-PAGE gel (Invitrogen Corporation, CA) using MES buffer and transferred onto nitrocellulose membrane. Membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 60 min and incubated overnight at 4 °C with the appropriate primary antibody in 5% milk in TBST. After washings with TBST, the membrane was incubated with the appropriate secondary antibody (Southern Biotech, AL) at room temperature for 2 h. After washing again with TBST, the membranes were developed using ECL plus (Millipore Corporation, MA) and the image was captured using alpha-imager Fluoretech HD2. The following antibodies were used for Western blotting analysis: AnxA2 (BD Biosciences, CA), TSG101 (BD Biosciences, CA), flotillin-1 (BD Biosciences, CA), calnexin (BD Biosciences, CA), GM130 (BD Biosciences, CA), EpCAM (Cell Signaling Technology, MA), and CD9 (Cell Signaling Technology, MA).

HeLa cells were fixed with 4% paraformaldehyde (PFA)/PBS and permeabilized with 0.25% Triton X-100. The following antibodies were used: GCN1 (Abcam, Cambridge, UK), calnexin (BD Biosciences, Tokyo, Japan), pyruvate dehydrogenase (PDH) subunit E1 antibody (Invitrogen, Carlsbad, CA). Detection was performed using Alexa Fluor 488 for GCN1 and Alexa Fluor 594 for calnexin and PDH. 4,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. The fluorescent images were observed using the C1si confocal imaging system (Nikon, Tokyo, Japan).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate equal protein amounts of mEVs and whey samples. Proteins were transferred onto polyvinylidene fluoride membranes, blocked with 5% skim milk (BD Biosciences, Franklin Lakes NJ, USA), and detected with the following primary antibodies: HSP70, TSG101, CD63, CD9 and calnexin (cat. no. ab275018, Abcam, UK). After incubation, the membranes were washed by TBST, and then incubated for 50 min at 37 °C with secondary antibody goat anti-rabbit IgG (ZSGB-bio, Beijing, China). The protein bands were washed by TBST and scanned with an automatic ECL image analysis system (Tanon-4800, Shanghai, China). In addition, the antibodies to verify globe proteome were ABAT (ab216465), SCIN (ab199723), DDC (ab131282), ACSL4 (ab155282) and GAPDH (ab9485), purchased from Abcam UK (Abcam, Cambridge, UK).

Ea.Hy-926 cell pellets were directly lysed in 2× SDS sample buffer and then incubated at 95°C for 10 min to ensure complete lysis. Lysates were separated on 6.5–15% gradient cells and transferred to PVDF membranes. Membranes were blocked in 5% milk and incubated overnight with the respective primary antibodies. The following day, membranes were washed, incubated with secondary antibodies, washed again and developed using ECL.
Primary antibodies used were: GAPDH (polyclonal rabbit (pcRb), Santa Cruz Biotechnology), β-Aktin (monoclonal mouse (mcM), C4, Santa Cruz Biotechnology), TCF11/Nrf1 (mcRb, D5B10, Cell Signalling), DDI2 (pcRb, ab197081, Abcam), p97/VCP (pcRb, Lab Stock), PSMA7/α4 (pcRb, Lab Stock), PSMA1/α6 (mcM, MCP20, Enzo Life Sciences), Tubulin (mcM, Covance Antibody Products), Calnexin (mcM, BD Biosciences), Lamin B1 (mcM, Invitrogen), CAPNS1 (pcRb, GeneTex). Secondary antibodies used were anti-mouse HRP and anti-rabbit HRP (Calbiochem).