Figure 5. IL-23 induces the formation of p-STAT3/p-STAT4 heterodimers.

Splenocytes from naive Vα2.3/Vβ8.2 TCR Tg mice were transfected with either si-NS or si-Stat4 for 18 hours. (A) Knockdown of STAT4 was determined by Western blot of si-NS and si-STAT4 transfected cells using tubulin as the control protein. (B) The si-STAT4 or si-NS transfected cells were activated with anti-CD3/CD28 plus IL-6+IL-23 for 60 hours and then adoptively transferred into naive B10.PL mice (5 × 10⁶ cells per mouse). The number of mice with clinical signs/total number of mice in each group is shown as follows: si-NS (7/10) and si-STAT4 (4/10). (C) Supernatants from anti-CD3/CD28 plus IL-6+IL-23 culture condition were analyzed by ELISA for IFN-γ, IL-17, and GM-CSF. *P < 0.05 (Mann-Whitney U test). (D) Purified CD4⁺ T cells from naive B10.PL mice were activated in vitro with anti-CD3/CD28 in the presence of the combinations of IL-6+IL-23 or IL-12+IL-23 for 64 hours. CD4⁺ T cells from naive mice served as a negative control. The 3 groups of cells were washed and rested in fresh medium for 4 hours, and then IL-23 or PBS were added as secondary stimulations for 30 minutes. Nuclear protein was extracted for use in a co-IP assay performed with Ab for total STAT3 or IgG control. Precipitated protein complexes and inputs were electrophoresed with SDS-PAGE. p-STAT4, p-STAT3, and total STAT3 were analyzed sequentially by Western blot, and images shown are from the same blot. (E) Splenocytes from naive Vα2.3/Vβ8.2 TCR Tg mice were activated with MBP Ac1-11 with and without IL-23 for 64 hours, and then CD4⁺ T cells were purified. Nonactivated T cells were the negative control. Nuclear protein was extracted for use in a co-IP assay performed with Ab for total STAT3 and an isotype control. Precipitated protein complexes and inputs were electrophoresed with SDS-PAGE. p-STAT4 (top) and p-STAT3 (bottom) were detected by Western blot.