RAW264.7 were treated with medium, irisin (400 ng/ml), or LPS (1000 ng/ml) for 24h, respectively, or after pretreatment for 12h, LPS was added for 12h, and then were collected to determine the expression of ERK, pERK, AKT, pAKT, PPAR α, and PPAR γ by Western blot assay described as the “Materials and Methods” section. A) Influence of irisin and LPS on the protein expression levels of AKT, pAKT, and densitometric quantification normalized to GAPDH. B) Influence of irisin and LPS on the protein expression levels of ERK, pERK, and densitometric quantification normalized to GAPDH. C) Influence of irisin and LPS on the protein expression levels of PPAR α, PPAR γ and densitometric quantification normalized to GAPDH. D) for cytokine protein determination, RAW264.7 were treated with medium, irisin (400ng/ml), and LPS (1000ng/ml) for 24h respectively, or after pretreatment with irisin for 12h, LPS was added for 12h, then cell-free supernatants were collected and assessed for IL-12 and IL-23 production by ELISA. Data presented as mean ± SE (n=3). *P<0.05, *** P<0.001, **** P< 0.0001. Control, medium; Irisin, 400ng/ml irisin; LPS, 1000 ng/ml LPS; Irisin + LPS: after pretreatment with irisin (400 ng/ml) for 12h, LPS (1000 ng/ml) was added for 12h.