Phospho stat3 tyr705 rabbit mab d3a7

PBMCs were lysed on ice for 30 min using RIPA lysis buffer (Beyotime) with protease inhibitors (Beyotime) and phosphatase inhibitors cocktail (Beyotime), and the protein concentration was determined by a bicinchoninic acid assay kit (Biosharp). The proteins (50 µg per lane) were mixed with one-fifth volume of 5× loading buffer, separated by 10% SDS-PAGE, and were then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h with 5% nonfat milk at room temperature and were incubated overnight at 4°C with primary antibodies against STAT3 rabbit mAb (79D7, Cell Signaling Technology), phospho-STAT3-Tyr₇₀₅ rabbit mAb (D3A7, Cell Signaling Technology), NF-κB p65 rabbit mAb (D14E12, Cell Signaling Technology), anti-NF-kB p65-phospho-S₅₃₆ antibody (EP2294Y, Abcam), and β-actin rabbit monoclonal antibody (Beyotime). After 3 washes with Tris-buffered saline with 0.1% Tween® 20 detergent, the membranes were incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibody (GE) and were detected with enhanced chemiluminescence (Beyotime). The results were analyzed using Image Studio software.