Western Blotting for Protein Quantification

Western blotting was performed as described following the previous method [21 (link)]. Cells (1 × 10⁶ cells/well) were harvested and homogenized in the radioimmunoprecipitation assay cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins (80 μg) were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel. Subsequently, resolved proteins were transferred to nitrocellulose membranes. Membranes were blocked in 5% BSA for one hour. The blots were washed with Tris-buffered saline with Tween 20 buffer (TBST) and incubated with primary antibodies: phosphorylated-Syk (Cell Signaling Technology), phosphorylated-Gab (Cell Signaling Technology), phosphorylated c-Jun (Cell Signaling Technology), and β-actin (Cell Signaling Technology). The immunoblots were washed in TBST(Bio-Rad Laboratories, Hercules, CA, USA) and incubated with horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology). The immuno blots were developed using EZ west Lumi plus (Atto, Tokyo, Japan). Finally, developed immuno blots were visualized, and a densitometry analysis of the bands was performed using LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE, USA).