Horseradish peroxidase linked goat anti rabbit secondary antibody

Exosome and cellular proteins were obtained by fractionation of cell lysates with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The protein bands were transferred onto a polyvinylidene fluoride membrane and analyzed using rabbit monoclonal anti-CD9 (Abcam, Cambridge, UK), anti-CD63 (Abcam), anti-p44/42 mitogen-activation protein kinase (MAPK; Thr202/204; Cell Signaling, Danvers, MA, USA), polyclonal anti-phospho p44/42 MAPK (Cell Signaling), and anti-β-actin antibodies (Abcam) at 4 °C overnight. Before probing, nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBST (10 mM Tris, pH 8.0, 150 mM sodium chloride (NaCl), and 0.5% Tween-20) for 60 min at room temperature. Membranes were washed four times for 10 min each and incubated with horseradish peroxidase-linked goat anti-rabbit secondary antibody (1:3000; Abcam) at room temperature for 1 h. Blots were washed four times with TBST and developed with the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Waltham, MA, USA) according to the manufacturer’s protocols and were quantified by using Image J (Version 1.50, National Institutes of Health, Bethesda, MD, USA).

Exosome and cellular proteins were obtained by fractionation of cell lysates with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The protein bands were transferred onto a polyvinylidene fluoride membrane (PVDF) and incubated overnight at 4 °C with the following antibodies: rabbit monoclonal anti-CD9 (Abcam, Cambridge, UK), anti-CD63 (Abcam), anti-calnexin (Abcam), anti-CD81 (Invitrogen, MA, USA), anti-TSG101 (Invitrogen), and anti-GM130 (Cell Signaling, MA, USA). Before probing, nonspecific binding was blocked by incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) for 60 min at room temperature. Membranes were washed four times for 10 min each and incubated with horseradish peroxidase-linked goat anti-rabbit secondary antibody (1:3000; Abcam) at room temperature for 1 h. Blots were washed four times with TBST and developed with the enhanced chemiluminescence system (Amersham Biosciences, Waltham, MA, USA) according to the manufacturer’s protocols and were visualized using the Chemidoc imaging system (BioRad, Hercules, CA, USA).