Phosphorylated syk

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).

Protein analysis was performed as previously described [13 (link),18 (link)]. Proteins (80 µg) from each sample were electrophoresed on 7–10% gradient polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were blocked in 5% BSA for 1 h and incubated with primary antibodies against phosphorylated Syk (Cell Signaling Technology), phosphorylated LAT (Cell Signaling Technology), phosphorylated ERK (Cell Signaling Technology), phosphorylated NF-κB (Cell Signaling Technology), phosphorylated ERK1/2 (Cell Signaling Technology), and β-actin (Cell Signaling Technology). After washing with Tris-buffered saline with Tween 20 (TBST) buffer (Bio-Rad Laboratories, Hercules, CA, USA), the membranes were incubated with horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology). The bands were detected using LI-COR Odyssey (LI-COR Biosciences).