Amersham ecl plus western blot detection system

MSCTRAIL cells or EVs were lysed in RIPA buffer for total protein extraction and the protein concentration in the lysate was determined using a BCA protein assay. 30 µg of cellular lysate proteins or 20 µg of EV proteins for each sample were resolved on 4–12% polyacrylamide sodium dodecyl sulphate gels and analysed by means of immunoblotting with primary rabbit anti-human TRAIL (c-terminal) (ab42121, Abcam) antibody and primary mouse anti-CD9, anti-CD81 and anti-CD63 antibodies (Invitrogen, UK). An HRP-conjugated secondary antibody against rabbit or mouse IgG (Cell Signaling Technology, UK) was used accordingly. Signals were detected with the Amersham ECL plus western blot detection system (GE Healthcare).

THP-1 monocytic cells, cultured in RPMI-1640 complete medium at a cell density of 10⁶ cells per mL in triplicate wells, were treated with H₂O₂ (10 mM) for 30 min and/or TNF-α (10 ng/mL) for 10 min, unless stated otherwise, in a humidified incubator (5% CO₂), while BSA-treated cells served as vehicle control. Harvested cells were incubated for 30 min with cell lysis buffer containing Tris 62.5 mM (pH 7.5), 1% Triton X-100, and 10% glycerol, lysates were clarified by centrifugation at 10,000× g for 10 min and supernatants were collected. Protein concentration was measured using Quickstart Bradford Dye (cat# 5000205, Bio-Rad Laboratories, Hercules, CA, USA). Cell lysates were resolved using 12% SDS-PAGE. Blots were probed with rabbit anti-human antibodies (1:1000 dilution) against phospho-NF-κB, total-NF-κB, phospho-ERK1/2, total-ERK1/2, phospho-IκBα, total-IκBα and beta actin at 4 °C overnight. All primary antibodies were purchased from Cell Signaling (CST Inc., Danvers, MA, USA). Blots were washed with TBS 3 times and incubated with HRP-conjugated secondary antibody (1:2500 dilution, Promega, Madison, WI, USA) for 2 h. Immunoreactive bands were developed by using Amersham ECL Plus Western Blot Detection System (GE Health Care, Buckinghamshire, UK) and visualized by ImageDoc™ MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA).

AKT inhibitor treated cell lysates were obtained using RIPA lysis buffer supplemented with protease and phosphatase inhibitor tablets (Roche). Protein samples (30 µg/lane) were run by SDS-PAGE and transferred to Hybond C extra nitrocellulose membranes (Amersham Biosciences) before incubation with the following primary antibodies; pAKT, total AKT, pGSK3β, total GSK3 β, p(Cell Signaling Technologies), Actin (Sigma) or Vinculin (Santa Cruz). Primary antibodies were diluted at 1:1000 (except Actin which was diluted 1:5000). Secondary antibodies were diluted 1:10,000. Blots were visualised using Amersham ECL plus western blot detection system (GE Healthcare) or Odyssey CLx system (LI-COR).