Fig. 4: IGFBP7 impedes ubiquitination and subsequent degradation of STAT3 mediated by the E3 ubiquitin ligase MDM2. | Nature Communications

Fig. 4: IGFBP7 impedes ubiquitination and subsequent degradation of STAT3 mediated by the E3 ubiquitin ligase MDM2.

From: Renal insulin-like growth factor binding-protein 7 is a critical promoter of progressive diabetic kidney disease

Fig. 4: IGFBP7 impedes ubiquitination and subsequent degradation of STAT3 mediated by the E3 ubiquitin ligase MDM2.

A Venn diagram of the high glucose (HG) model group and IGFBP7 overexpression (OE) group. Filtering the binding strength with differentially expressed protein in high glucose-stimulated mTEC cells with or without IGFBP7, identified STAT3 as a direct target protein of IGFBP7. B Co-IP results of IGFBP7 binding to STAT3 in control and high glucose model groups in renal tubular epithelial cells. C Co-IP results of HA-IGFBP7 binding to Flag-STAT3 in HEK-293T cells. D Surface plasmon resonance (SPR) analysis of the interactions between recombinant STAT3 protein and recombinant IGFBP7 protein. Recombinant IGFBP7 protein concentration: 25, 12.5, 6.25, 3.125, and 1.56 μM. E Immunofluorescence staining of IGFBP7 and STAT3 in HG-treated mTEC and HK2 cells. F Molecular docking of IGFBP7 with STAT3. G Co-IP was performed to determine the interaction between endogenous STAT3 and the functional domains of IGFBP7, including Flag-IGFBP7, Flag-Nter, Flag-Nter-Ka, Flag-Ka-Ig, and Flag-Ig in HG-treated HK2 cells. H Co-IP was performed to determine the interaction between endogenous IGFBP7 and the functional domains of STAT3, including Flag-STAT3, Flag-CCD, Flag-DBD, and Flag-SH2-TAD in HG-treated HK2 cells. I Western blotting analysis of STAT3 levels in HG-treated mTEC and HK2 cells with or without IGFBP7 overexpression (n = 4). J The UbiBrowser assay results indicate that MDM2 is the key E3 ubiquitin ligase for STAT3. K Co-IP assay detects the effect of MDM2 overexpressing on STAT3 ubiquitination level in HK2 cells. L Flag-STAT3, Myc-MDM2, and IGFBP7 OE were co-expressed and applied with MG-132 in HK2 cells. Flag was isolated by IP, and levels were assessed with anti-Myc antibody. M Myc-MDM2, Flag-STAT3, and IGFBP7 OE were co-expressed and applied with MG-132 in HK2 cells. Myc was isolated by IP, and levels were assessed with anti-Flag antibody. Data represent the mean ± SEM. mTEC mouse renal tubular epithelial cells, HK2 human renal tubular epithelial cells, LC-MS/MS liquid chromatography-tandem mass spectrometry, EV empty vector, OE overexpression, Nter IGFBP N-terminal, KA Kazal-like, Ig Ig-like C2-type, CCD Coiled-coil domain, DBD DNA-binding domain, SH2-TAD Src homology 2-transactivation domain. Source data are provided as a Source Data file.

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