Figure 4
CBD modulates Th17 signaling pathways in T MOG cells co-cultured with spleen-derived APC. Pre-adhered APC were co-cultured with TMOG cells and stimulated with MOG35-55 in the presence or absence of CBD. CD4+ microbead purified TMOG cells were lysed and proteins subjected to gel electrophoresis, followed by transfer to nitrocellulose membrane and immunostaining of the phosphorylated forms of Akt, STAT3, and STAT5 as well as of total amount of β-actin and STAT3. The bar graphs show average percentage values (in comparison to MOG35-55 without CBD as 100%) of (A) phospho-STAT3 Tyr705 (ANOVA F(3,4) = 94.3, P < 0.001); (B) phospho-STAT3 Ser727 (ANOVA F(3,4) = 96.7, P < 0.001), (C) phospho-Akt (ANOVA F(3,4) = 96.7, P < 0.001); (D) phospho-STAT5 (ANOVA F(3,7) = 32.6, P < 0.001); (E) representative blots showing the phosphorylated forms of Akt, STAT3 (Tyr705 and Ser727), and STAT5 Tyr694 as well as total levels of β-actin and STAT3 (n = 2 to 3); (F) in this control experiment, resting TMOG cells were cultured without APC and without MOG35-55 for 8 h in the presence or absence of 5 μM CBD in maintenance medium. Representative immunoblots (n = 2) show that CBD does not affect the levels of phospho-Akt, phospho-STAT3, and phospho-STAT5 in resting TMOG cells cultured without APC. Symbols: *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated purified TMOG; ## P < 0.001, ### P < 0.001 vs MOG35-55-treated TMOG cells.