3.4. Effects of MQL on MAP kinase signaling pathways
Next, to investigate MAP kinases upstream of MQL-induced HO-1 expression, we cultured CD4+ T cells with U0126 (ERK inhibitor), SB20350 (p38 inhibitor), or SP600125 (JNK inhibitor) in the presence of MQL. As a result, MQL-induced HO-1 expression was most significantly reduced by U0126 treatment compared with the other inhibitors (Fig. 4A) . To explore this further, we measured ERK1/2, p38, and JNK activation in the presence or absence of T cell stimulation (anti-CD3/CD28 antibodies). Consistent with the previous finding, MQL dose-dependently increased the phosphorylation of ERK, but not p38 or JNK (Fig. 4B and C) .
The Ras-Raf-MEK-ERK signaling pathway plays a crucial role in gene expression related to cell growth, proliferation, differentiation, and survival30,31. Ras proteins are small GTPases that can activate Raf-MEK-ERK signaling32. GTP-bound Ras interacts with the Raf family (A-Raf, B-Raf, and C-Raf), inducing Raf dimerization and phosphorylation of C-Raf (also called Raf-1)33,34. It has also been reported that phosphorylation of C-Raf (Ser338) can induce ERK activation in lymphocytes such as T cells and B cells35. Therefore, we hypothesized that C-Raf could be the signal upstream of ERK, and indeed found that MQL treatment induced phosphorylation of C-Raf (Ser338) (Fig. 4D) .