Ab155539

The mouse anti-CI-MPR antibody (Ab2733; Abcam) and rabbit anti-CI-MPR antibody (ab124767; Abcam) were used for immunofluorescence and Western blot, respectively. The antibodies for Giantin (924,302; Convance), HRS (ab155539; Abcam), GM130 (610,822; BD Biosciences) and EEA1 (610,456; BD Biosciences) were used for immunofluorescence. The antibodies for γ1 (610,386; BD Biosciences), γ2 (HPA004106; Sigma-Aldrich), μ1A (AB111135; Abcam), GAPDH (G9545; Sigma-Aldrich) and β-actin (sc-47778; Santa Cruz) were used for Western blot. The same antibodies for γ1 and γ2 were also used for immunofluorescence. The rabbit anti-Vps26 and anti-Snx2 anti-serum were kind gifts from Juan Bonifacino, and previously described (97 (link), 98 (link)). The rabbit anti-GFP antibody used for western-blot was a gift from R. Hegde (MRC). The mouse monoclonal anti-CD8 antibody used for flow cytometry and antibody uptake assays was a kind gift from Matthew Seaman (MRC) (9 (link)). Horseradish-peroxidase-conjugated donkey anti-mouse immunoglobulin G (IgG) and donkey anti-rabbit IgG were obtained from GE Healthcare. Secondary antibodies conjugated to Alexa fluorophores were purchased from Thermo Scientific.

The antibodies used for immunofluorescence (IF) were rabbit polyclonal anti-TGN46 (ABT95; Millipore), sheep polyclonal anti-TGN46 (Abd Serotec AHP500; Bio-Rad), rabbit polyclonal anti-SNX2 and anti-VPS26 (gifts from C. Haft, NIDDK, NIH) (43 (link)), Alexa Fluor 488-labeled mouse monoclonal antibody (MAb) anti-HRSV F protein (133-1H MAB8262X; Millipore), mouse MAb anti-HRSV N protein labeled with FITC (MAB858-3F N; Millipore), mouse polyclonal anti-HRSV M protein (44 (link)), rabbit polyclonal anti-giantin (PRB-114C-200; Covance), mouse MAb anti-RSV F protein (MAB8599; Millipore), rabbit polyclonal anti-Lamp-1 D2D11 XPR (9091T; Cell Signaling), mouse MAb anti-EEA1 (610457; BD), rabbit polyclonal HRS (ab155539), Alexa Fluor 594-labeled goat anti-rabbit (ab150080; Abcam), Alexa Fluor 647-labeled goat anti-mouse (ab150115; Abcam), Alexa Fluor 488-labeled donkey anti-mouse (A21202; Invitrogen), and Alexa Fluor 647-labeled donkey anti-sheep (A21448 Thermo Fisher).

RIPA lysate, including a protease inhibitor, was applied to extract the total proteins from treated HepG2 cells or exosomes. After centrifugation at 4°C, the supernatant was taken. After the protein concentration was determined by the BCA method, the total protein (40 μg) in each group was separated by 12% SDS-PAGE electrophoresis and transferred into PVDF membranes. Then the target protein in membrane was sealed with 5% skim milk, and addressed with primary antibodies at 4°C overnight and the corresponding secondary antibodies for 2 h. After the reaction of the ECL reagent (Bio-Rad) for 5 min, the results were obtained by developing imaging in a dark room. And the primary antibodies contained CD63 (1 : 5,000, ab134045, Abcam), CD9 (1 : 2000, ab92726, Abcam), TSG101 (1 : 5,000, ab125011, Abcam), Calnexin-CNX (1 : 20,000, ab92573, Abcam), cleaved caspase-3 (1 : 500, ab32042, Abcam), Cyclin D1 (1 : 10000, ab134175, Abcam), Alix (1 : 1000, ab275377, Abcam), Hrs (1 : 1000, ab155539, Abcam), Rab27A (1 : 3000, ab55667, Abcam), Vps4A (1 : 3000, ab229806, Abcam), BAG5 (1 : 2000, ab97660, Abcam), TRPV6 (1 : 400, 39563, SAB), and GAPDH (1 : 10000, ab8245, Abcam).