Protein quanti cation kit rapid

Tissue and HUVECs were homogenized in ice-cold Piece RIPA Buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl uoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quanti cation Kit-Rapid (Dojindo Laboratories). Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63(Abcam), CD9(Abcam), CD81(Cell Signaling Technology, Inc. Danvers, MA, USA), βactin (Cell Signaling Technology), phospho-Akt (Ser473) (Cell Signaling Technology), and Akt (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) Substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).

Tissue and HUVECs were homogenized in ice-cold Pierce RIPA Buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl uoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quanti cation Kit-Rapid (Dojindo Laboratories).
Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63 (Abcam), CD9 (Abcam), CD81 (Cell Signaling Technology, Inc. Danvers, MA, USA), βactin (Cell Signaling Technology), phospho-Akt (Ser473)(Cell Signaling Technology), Akt (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), and caspase-3 (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).