Western Blot THP-1 Culture

Cell pellets from THP-1 cells were suspended on ice-cold lysis buffer [phosphate buffer saline (PBS) containing 2% sodium dodecyl sulfate (SDS) and complete protease inhibitor cocktail (Roche, catalog 11697498001)]. Protein concentration of cell extracts was determined using the bicinchoninic acid (BCA) protein kit (Pierce, catalog 23225). Proteins were separated on 10% SDS-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane. After 1 h of blocking with 5% fat-free milk in Tris-Tween-Buffer-Saline, the membranes were probed for 1 h at room temperature with mouse monoclonal TRAFD1 antibody 1:1,000 (Invitrogen, catalog 8E6E7) or mouse monoclonal anti-actin antibody 1:5,000 (MP Biomedicals, catalog 08691001), followed by incubation with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies 1:10,000 (Jackson ImmunoResearch, catalog 115-035-003). After three 10-min washes, the bands were detected by Lumi-Light Western blot (WB) substrate (Roche, catalog 12015200001) in a Chemidoc MP imaging system (Bio-Rad) and quantified using Image Lab™ software (Bio-Rad). The band intensity of TRAFD1 was normalized to actin, and the TRAFD1 SCR control level was set as 100%.