Fig 1: Cinacalcet suppresses the PKCd/ERK/P65 signaling pathway. (A) Visualization of predicted the binding sites of cinacalcet with respect to NK1R. Structures on the yellow color and grey surface represent the NK1R structure, blue structures represent the amino acids of cinacalcet that bind to NK1R, and structures in pink represent structures of cinacalcet. (B) DARTs assay to test the binding of NK1R and cinacalcet. (C) Representative western blot results to show that cinacalcet suppresses the PKCd/ERK/P65 signaling pathway. Bone marrow-derived macrophages (BMDMs) were starved with 2% FBS overnight. After treating the cells with cinacalcet (1 µM) for 2 h, TNFa (10 ng/ml) was added for different periods (0, 15, 30, 60 min). Proteins were extracted from the cells, and western blotting was performed. (D) Quantification of p-PKCd expression. (E) Quantification of p-ERK expression. (F) Quantification of p-P65 expression. (G, H) qRT-PCR was performed to test the mRNA expression levels of IL-1ß and IL-6 in BMDMs after stimulation with TNFa (10 ng/ml) in the presence or absence of cinacalcet for 24 h (I, J) ELISA was performed to detect IL-1ß and IL-6 levels in BMDM supernatants after stimulation with TNFa (10 ng/ml) in the presence or absence of cinacalcet for 24 h. (K) Proposed model explaining the anti-TNF activity of cinacalcet through direct targeting of the NK1R pathway. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are shown as the mean with standard deviation. Three independent experiments were performed.