Fig. 6: TNFAIP3 was a direct binding partner of ITGBL1 in NF-κB signal-mediated fibroblast activation.

a The schematic procedure of probing the binding partners of ITGBL1 in fibroblasts by Co-IP and LC-MS/MS analysis methods. b Co-IP, LC-MS/MS, and cluster analysis of the binding proteins for targeted ITGBL1 protein using the ITGBL1 antibody (samples 4, 5, and 6) or control IgG (sample 1, 2, and 3) in ITGBL1-overexpressing WI-38 cells. Red represents high scores and green represents low scores. The color brightness of each unit is associated with differences in multiples (log 2[AR/N]). c, d Co-IP in combination with western blot performed to validate the interaction between ITGBL1 protein and TNFAIP3 protein in ITGBL1-overexpressing WI-38 and LX-2 cells, or control cells. e, f Immunofluorescence detection of the co-location of ITGBL1 and TNFAIP3 in ITGBL1-overexpressing WI-38 and LX-2 cells, or control cells. Scale bar, 50 μm. g–j Western blot and quantitative assay of TNFAIP3, p-IKKα/β, and p-p65 in TNFAIP3-silent or/and ITGBL1-overexpressing WI-38 and LX-2 cells, or control cells. Figure 4i, j was the quantitative results for Fig. 4g, h, respectively. k Relative luciferase activity of NF-κB in TNFAIP3-silent or ITGBL1-overexpressing WI-38 and LX-2 cells, or control cells. Each experiment was performed at least in triplicate and all the data are shown as mean ± SD. Student’s t-test was used to analyze the data; *p < 0.05, **p < 0.01.