Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Ischemia-reperfusion injury elicits time-dependent changes of TECs injury, inflammatory infiltration, and interstitial fibrotic remodeling. A , schematic illustration of the experimental protocol for inducing renal IRI in a mouse model. B , levels of Scr from sham-operated mice and mice sacrificed at 1 h, 1 day, 3 days, 7 days, 28 days after I/R. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Havcr1/Kim1 in renal cortical samples. E , immunohistochemical detection of Havcr1/Kim1 expression in renal cortical sections from sham and I/R-treated mice. The scale bar represents 100 μm. F and G , protein expression profiling by western blot ( F ) and subsequent statistical analysis ( G ) of IL-6 in renal cortical samples. H , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections. The scale bar represents 40 μm. I , histopathological assessment of renal tissue architecture (H&E) and collagen deposition (Masson’s trichrome) in experimental groups. The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; Scr, serum creatinine; TEC, tubular epithelial cell.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Marker, Virus

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Dock family proteins are identified, with knockdown of Dock2 demonstrating a significant attenuation of the proinflammatory response of TECs in vitro . A , heatmap of Dock family proteins and phosphosites (ANOVA, p < 0.05). B , intensity plots showing Dock2 ( black line ) and its phosphosite S1704 (Dock2_pS1704, green line ) across the six experimental groups. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Dock2 in renal cortical samples. E , immunofluorescent detection of Dock2 and Havcr1/Kim1 expression and colocalization in renal tissue sections from sham and I/R-treated mice. The scale bar represents 40 μm. F , the knockdown efficiency of Dock2 by siRNA in H/R-treated HK-2 cells was analyzed by western blot. G , the statistical analysis of ( F ). H – J , RT-qPCR showing the effects of Dock2 knockdown on mRNA expression levels of MCP-1, TNF-α, and IL-6. K , western blot analysis was performed to assess the effects of Dock2 knockdown on IKK-β phosphorylation in H/R-treated HK-2 cells. L , densitometric analysis of p-IKKβ from ( K ) was performed with normalization to the respective total protein. M , translocation of NF-κB p65 in HK-2 cells was detected by immunofluorescence. NF-κB p65 ( green ), DAPI ( blue ). The scale bar represents 5 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; Dock2, dedicator of cytokinesis 2; Havcr, hepatitis A virus cellular receptor 1; H/R, hypoxia/reoxygenation; IKKβ, IκB kinase beta; Kim1, kidney injury molecule-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; RT-qPCR, reverse transcription quantitative PCR; TEC, tubular epithelial cell; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Knockdown, In Vitro, Phospho-proteomics, Expressing, Western Blot, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Inhibition of Dock2 by CPYPP attenuates tubular injury, inflammatory infiltration and interstitial fibrosis after renal IRI in mice. A , schematic of the experimental design for unilateral I/R in mice treated with CPYPP or vehicle via i.p. injection following I/R induction. B , measurement of Scr levels. C , H&E staining was performed to assess the effect of pharmacological inhibition of Dock2 by CPYPP on renal tissue architecture in mice at 3 days and 28 days post-I/R. Inflammatory cells, tubular casts, and dilated tubules are indicated by solid arrows , hollow arrows , and triangles , respectively. The scale bar represents 100 μm. D , tubular injury score at 3 days post-I/R (quantified from C ). E – I , western blot ( E ) and quantitative analysis ( F – I ) of renal cortical Havcr1/Kim1, IL-6, TNF-α, and MCP-1 expression in sham-operated and 3 days post-I/R mice, with or without CPYPP treatment. J , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections to assess inflammatory infiltration. The scale bar represents 40 μm. K , the quantitative analysis of the F4/80 immunofluorescence staining from ( J ). L – N , western blot ( L ) and quantitative analysis ( M and N ) of renal cortical FN and α-SMA expression in sham-operated and 28 days post-I/R mice, with or without CPYPP treatment. O , Masson's trichrome staining was used to evaluate the effect of CPYPP on renal collagen deposition at 28 days post-I/R, with quantification data shown in ( P ). The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. CPYPP, 4-[3′-(2″-chlorophenyl)-2′-propen-1′-ylidene]-1-phenyl-3,5-pyrazolidinedione; Dock2, dedicator of cytokinesis 2; FN, fibronectin; Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; MCP-1, monocyte chemoattractant protein-1; α-SMA, alpha-smooth muscle actin; Scr, serum creatinine; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Inhibition, Injection, Staining, Western Blot, Expressing, Marker, Immunofluorescence, Virus