Can get signal pvdf blocking reagent

EV pellets were lysed in RIPA assay buffer (FUJIFILM Wako Pure Chemical Corporation) for 30 min on ice. Protein samples were electrophoresed on a 10% SDS–polyacrylamide gel and blotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were washed with TBS-Tween and were shaken in Can Get Signal PVDF Blocking Reagent (TOYOBO) at room temperature for 1 h. After washed, the membranes were incubated with primary antibodies diluted in Can Get Signal Solution 1 (TOYOBO) at room temperature for 1 h. After washed, the membrane was incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) diluted in Can Get Signal Solution 2 (TOYOBO) at room temperature for 1 h. After washed, the membranes were incubated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, KGaA, Darmstadt, Germany) for a few seconds, and chemiluminescence was detected using the ChemiDoc Touch system (Bio-Rad). The following antibodies were used: mouse anti-CD63 (Santa Cruz Biotechnology, Santa Cruz, CA) and sheep anti-mouse IgG HRP (Sigma).

The cells were lysed in a 1× RIPA Lysis Buffer (EMD Millipore, Burlington, MA) containing a complete Protease Inhibitor Cocktail and PhosSTOP (Roche Applied Science, Penzberg, Germany). Proteins were electrophoresed on 4%–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA) and transferred to membranes using a Trans-Blot Turbo Transfer Pack PVDF (Mini) (Bio-Rad). The membranes were blocked with Can Get Signal/PVDF Blocking Reagent (Toyobo, Osaka, Japan) and incubated with primary antibodies overnight using Can Get Signal Solution 1 (Toyobo). After washing three times with Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (Cell Signaling Technology, Danvers, MA) for 1 hour. The membranes were imaged by ChemiDoc XRS Plus (Bio-Rad). The following primary antibodies were used: phosphorylated (p)-epidermal growth factor receptor (EGFR; Tyr1068) (D7A5), EGFR (D38B1), phosphorylated (p)-IGF-I receptor β (Tyr1135/1136)/insulin receptor β (INSRβ; Tyr1150/1151) (19H7), IGF-I receptor β, p-AKT (Ser473), AKT (pan) (11E7), p-SHP-2 (Tyr542), SHP-2 (D50F2), β-actin, and GAPDH (Cell Signaling Technology).

Cells were harvested in a lysis buffer (100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 2% β-mercaptoethanol, and 0.4 mg/ml bromophenol blue), and boiled at 100 °C for 10 min. Soluble proteins (25 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad) by Trans-blot turbo (Bio-Rad). The membrane was immersed with the Can Get Signal PVDF blocking Reagent (Toyobo) for 1 h, and incubated with monoclonal antibodies against RSV-G protein (1:1000, 94966; Abcam, Cambridge, UK) and β-actin (1:5000, A5316; Sigma-Aldrich, St. Louis, Missouri, US) for overnight. After washing with PBS containing 0.1% tween 20, the membrane was blotted with horseradish peroxidase-conjugated secondary antibody followed by visualization with a chemiluminescence agent, ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, Illinois, US) by LAS-3000 (GE Healthcare).