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Detection of the expression of STAT3 and its phosphorylation

LL Lijie Li
ZL Zhouzhou Liao
MY Mingzhu Ye
JJ Jianfa Jiang
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The expression levels of STAT3 and p-STAT3 were measured using western blotting assay. Total protein was separated from DCs using RIPA lysis buffer. Then, the concentration of protein was determined using a BCA Protein Assay Reagent Kit. After that, 25 μg of protein was separated on 12% SDS-PAGE gel, and were transferred onto PVDF membranes. The membranes were then maintained with 5% non-fat milk for 1 h at room temperature followed by the anti-STAT3 and anti-p-STAT3 antibodies incubation overnight at 4 °C. Next day, the membranes were incubated with secondary goat anti-rabbit for 1 h at room temperature. At last, an enhanced chemiluminescence kit was utilized to determine the protein bands, and the optical density of the western blot was analyzed using the Image-Pro Plus 6.0 (Media Cybernetics, lnc., USA) software. The relative expression of STAT3 and p-STAT3 was normalized to β-actin.

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