Protein Immunoblotting Analysis Protocol

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).