Fig 2: Binding assy of mindin to TLR4 proteins by SPR. Different concen-trations of TLR4 protein (3.125, 6.25, 12.5, 25.0, 50.0, 100.0 nM) were performed to analyse the binding and dissociation rate constants between mindin and TLR4 proteins. The binding constant of them was calculated to evaluate protein binding

Fig 3: Mindin knockdown inhibited the activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. B–F Quantitative analysis of TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05, **P < 0.01 versus HR. G Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. ***P < 0.001 versus control HR group

Fig 4: Mindin overexpression promoted production of inflammatory mediators and activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A Representative photomicrographs showing the immunofluorescence of GFP in adGFP and adMindin cells. B Mindin protein expression in adGFP and adMindin cells. C–E Protein levels of TNF-α and MCP-1 in the media of cultured TECs by Western blot. *P < 0.05 versus adGFP Con or adMindn Con. #P < 0.05 versus adGFP HR. F TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in adGFP and adMindin cells induced by HR or not. G–J Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus adGFP Con or adMindn Con. #P < 0.05 versus adGFP HR. K Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in mindin-overexpressed HK-2 cells. ***P < 0.001 versus adGFP HR group

Fig 5: Mindin deficiency inhibited TLR4/JNK/NF-κB signaling activation in renal IR injury A TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in kidneys of WT and mindin KO mice induced by IR or not. B–E Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus WT Sham or KO Sham. #P < 0.05 versus WT IR