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. 2017 Sep 7;2(17):e91663. doi: 10.1172/jci.insight.91663

Figure 4. IL-23R signaling activates both STAT3 and STAT4.

Figure 4

Splenocytes from naive B10.PL mice were activated in vitro with anti-CD3/CD28 in the absence/presence of IL-23, IL-6, IL-12, or combinations for 66 hours. The cells were washed and rested in fresh medium for 4 hours and then were stimulated with either IL-23 or PBS for 30 minutes. (A and B) Cells were analyzed by flow cytometry. Gated CD4 T cells were analyzed for p-STAT3 and p-STAT4. (C) Whole cell lysate was extracted, and p-STAT3, STAT3, p-STAT4, STAT4, and β-actin levels were determined by Western blotting. (D and E) The band intensity of STAT3, STAT4, p-STAT3, and p-STAT4 were quantified by NIH ImageJ software, and no cytokine condition in the PBS group was used as reference for normalization. The ratio of phosphorylation was determined by comparing p-STAT3 to STAT3 and p-STAT4 to STAT4. The data is representative of 4 independent experiments. The a, b, and c in D and E illustrate qualitative changes in p-STAT4 (D) and p-STAT3 (E) in response to IL-23.