Anti calnexin antibody

Exosomes were lysed with a RIPA buffer, resuspended in the loading buffer, boiled at 95 °C for 5 min, and then electrophoresed on SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane, which was blocked with 5% non-fat dry milk in TBST. Immunodetection was performed with anti-HSP70 antibody(1:1000, Proteintech, China), anti-Flotillin-1 antibody (1:1000, Proteintech, China), anti-CD63 antibody (1:1000, Proteintech, China) and anti-calnexin antibody (1:1000, Proteintech, China) at a dilution of 1:1000 followed by incubation at 4 °C overnight. The next day, protein was incubated with appropriate HRP-conjugated secondary antibody (1:5,000, Abcam, USA). Bands were revealed using ECL Plus and then imaged on the electrophoresis gel imaging analysis system (D-Digital, USA) to analyze.

Cell culture medium was obtained from life technologies (Staley Rd Island, USA), and FBS was obtained from Pan seratech (Aidenbach, Bavaria, Germany). EDTA-free protein inhibitor cocktail was purchased from Bimake (Houston, USA), Polyethylenimine (PEI) were obtained from Polysciences (Warrington, PA).
The following primary antibodies were used: Anti-ANGPTL3 antibody (AF136, R&D Systems, Minneapolis, USA), Anti-His antibody (Catalog: M20008F, Abmart, Berkeley Heights, US), Anti-Beta actin antibody (Catalog: 20536-1-AP, Proteintech, Chicago, IL), Anti-Calnexin antibody (Catalog: 10427-2-AP, Proteintech, Chicago, IL). The following secondary antibodies were used: HRP conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), bovine anti-goat IgG (Proteintech, Chicago, IL).

Transfected cells grown on cover slips to 70%‐80% confluence were fixed in 4% paraformaldehyde, then underwent permeation using PBS with 0.5% Triton and were blocked with 3% FBS in PBS‐Triton. Cells were incubated with anti‐FLAG antibody (1:200, Abmart) and anti‐Calnexin antibody (1:50, Proteintech) overnight at 4°C. Alexa Fluorescence series secondary antibodies (Life Technologies, Oregon) were used. Finally, the cells were fixed in the ProLong™ Diamond Antifade Mountant (Life Technologies) and then photographed by a Leica SP8 confocal microscope.