Fig 1: CYP suppresses PAK1 activation by inhibiting Ser144 phosphorylation. (A) Computational docking analysis of CYP docking with PAK1 protein. (B and C) Biacore surface plasmon resonance analysis of the interaction between CYP and wildtype PAK1 (B) or the mutant PAK1 (S144A) (C). Note: A presents Human PAK1 while B presents Human PAK1 (S144A) mutant form. (D) Homogeneous time-resolved fluorescence assay assessing the inhibitory effect of CYP on PAK1 activity. (E) Western blot analysis of p-PAK1Ser144/PAK1 expression in DCs, Th2 and Th9 cells stimulated with OVA or IL-33 with/without CYP treatment. (F) Schematic diagram summarizing the underlying mechanisms: CYP attenuates dendritic cell maturation and IL-33 secretion by blocking the PAK1-JNK/NF-κB signaling, thereby suppressing Th2/Th9 differentiation. Concurrently, CYP directly inhibits naïve T cell differentiation into Th2/Th9 cells by blocking the IL-33 activated PAK1-JNK/NF-κB pathway. Overall, CYP alleviates OVA-induced allergic inflammation by targeting PAK1 to inhibit PAK1-JNK/NF-κB axis. CYP, α-Cyperone; KD, dissociation constant (lower value indicates stronger affinity) Ka, association rate constant (higher value indicates faster binding) Kd, dissociation rate constant (higher value indicates quicker dissociation); DCs, dendritic cells; OVA, ovalbumin; NF-κB, nuclear factor-κB; c-Jun, c-Jun N-terminal kinase (JNK)-mediated activated protein-1.