Goat anti rabbit igg antibody conjugated with horseradish peroxidase

Exosome samples (2 µg) were reduced by 100 mM dithiothreitol at 95°C for 3 min, subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and electrophoretically transferred to a polyvinylidene fluoride transfer membrane. The membrane was blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. In order to detect Alix, the membrane was probed with mouse anti-Alix antibody (1:20,000 dilution; BD Biosciences, San Jose, CA, USA) for 1 h at room temperature, and then allowed to react with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (1:2,000 dilution; Life Technologies, Grand Island, NY, USA) for 1 h at room temperature. In order to detect Hsp70, the membrane was probed with rabbit anti-Hsp70 antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, and then allowed to react with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was soaked with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA), and chemiluminescence was detected by a LAS-3000 instrument (FUJIFILM, Tokyo, Japan).

Three human HCC cell lines (PLC, HepG2, Huh-7) were purchased from the Korean Cell Line Bank (Seoul, Korea) and they were cultured in 90% Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 mg/L streptomycin (GIBCO) in a humidified atmosphere containing 5% CO2 at 37℃.
For immunoblotting, cells were lysed in radioimmune precipitation (RIPA) buffer (Upstate, NY, USA) supplemented with protease inhibitor. The cell lysates were electrophoresed on 10% polyacrylamide gel, transferred onto polyvinylidene difluoride membrane (Bio-Rad Laboratories, CA, USA) and blotted with appropriate primary and secondary antibodies. The signal was detected using ECL reagent kit (Biosciences, Buckinghamshire, UK) and exposed to an X-ray film. The primary antibodies used were rabbit anti-PGDH (1:5,000 dilution; Novus Biologicals, CO, USA), goat anti-COX-2 (1:500 dilution; Santa Cruz, CA, USA), and mouse anti-β-actin (1:10,000 dilution; Sigma, St. Louis, MO, USA). Goat anti-rabbit IgG antibody conjugated with horseradish peroxidase was used as the secondary antibody (Santa Cruz, CA, USA).