Figure 3. Phosphorylation of ribosomal S6 kinase (RSK), mitogen-activated protein kinase (MAPK), and S6 in human umbilical vein endothelial cells (HUVECs) and analysis of angiogenic factors secreted by MKN-28 cells and cancer-associated fibroblasts (CAFs) with or without itraconazole in vitro using proteome arrays. HUVECs (A) were treated with 1 or 100 μM itraconazole, and the phosphorylation of p44/42 MAPK, p90RSK, Akt, and S6 was analyzed by western blotting. A protein molecular weight marker was included in the first lane. MKN-28 cells (B): MKN-28 secreted interleukin-8 (IL-8), tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, chemokine (C-X-C motif) ligand (CXCL)13, and urokinase plasminogen activator receptor (uPAR), which may be associated with bevacizumab resistance in MKN-28 cells. vascular endothelial growth factor (VEGF) (box 6) was not detected. Itraconazole did not influence the secretion of VEGF by MKN-28 cells. Boxes: 1, CXCL13; 2, IL-8; 3, TIMP-1; 4, TIMP-2; and 5, uPAR. The other spots in the image are positive controls. CAFs (C): Representative images of Proteosome Profiler Assay membanes probed with conditioned medium collected from itraconazole- or saline-treated cells. A: Positive control, B: negative control, C: plasminogen activator inhibitor-1, D: TIMP-4, E: IL-1β, F: VEGF, and G: monocyte chemoattractant protein 1 (MCP-1). Itraconazole treatment dramatically suppressed the secretion of MCP-1 (D).