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

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: mRNA expression of ACE2, TMPRSS2, Kim‐1, and NRP‐1 in different types of cells from five human organ systems was analyzed by RT‐qPCR. (A−D) Relative mRNA expression levels of ACE2 (A), TMPRSS2 (B), Kim‐1 (C), and NRP‐1 (D) presented as relative C t values. β‐actin gene was used for normalization. Kim‐1, kidney injury molecule‐1; mRNA, messenger RNA; NRP‐1, neuropilin‐1; RT‐qPCR, real‐time quantitative polymerase chain reaction; TMPRSS2, transmembrane serine protease 2

Article Snippet: The primary antibodies used were a rabbit polyclonal antibody to ACE2 (ab15348, Abcam), rabbit monoclonal antibodies to TMPRSS2 (EPR3861) (ab92323, Abcam), Kim‐1 (E1R9N) (14971 S, CST) and NRP‐1 (EPR3113) (ab81321, Abcam), and a mouse monoclonal antibody to β‐tubulin.

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Blocking of infection by SARS‐CoV‐2 and SARS‐CoV pseudoviruses with S1, RBD, and other recombinant proteins of entry‐related factors in Huh‐7 cells. (A−E) Blocking of infection of SARS‐CoV‐2 and SARS‐CoV pseudoviruses by S1 (A), RBD (B), ACE2 (C), Kim‐1 (D), and NRP‐1 (E) recombinant proteins. ANOVA analysis was performed if multiple samples were involved. p‐value was calculated by unpaired two‐tailed Student's t test between uninfected cells and untreated infected cells, and between treated group and untreated infected group. p < 0.05 was considered as statistically significant. ANOVA, analysis of variance; RBD, receptor‐binding domain; Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SARS‐CoV, severe acute respiratory syndrome coronavirus

Article Snippet: The primary antibodies used were a rabbit polyclonal antibody to ACE2 (ab15348, Abcam), rabbit monoclonal antibodies to TMPRSS2 (EPR3861) (ab92323, Abcam), Kim‐1 (E1R9N) (14971 S, CST) and NRP‐1 (EPR3113) (ab81321, Abcam), and a mouse monoclonal antibody to β‐tubulin.

Techniques: Blocking Assay, Infection, Recombinant, Two Tailed Test, Binding Assay

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Analyses of the correlation between the infectivity of SARS‐CoV‐2 or SARS‐CoV and the protein expression levels of entry‐related factors. (A–J) Correlation of the infectivity of SARS‐CoV‐2 (A, C, E, G, and I) or SARS‐CoV (B, D, F, H, and J) with glycosylated ACE2 (gly‐ACE2) (A and B), unglycosylated ACE2 (ungly‐ACE2) (C and D), TMPRSS2 (E and F), Kim‐1 (G and H) and NRP‐1 (I and J). The “red” dots represent cell lines/types that were infected by the pseudoviruses. The “green” dots represent cell lines/types that were not infected by the pseudoviruses. Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SARS‐CoV, severe acute respiratory syndrome coronavirus; TMPRSS2, transmembrane serine protease 2

Article Snippet: The primary antibodies used were a rabbit polyclonal antibody to ACE2 (ab15348, Abcam), rabbit monoclonal antibodies to TMPRSS2 (EPR3861) (ab92323, Abcam), Kim‐1 (E1R9N) (14971 S, CST) and NRP‐1 (EPR3113) (ab81321, Abcam), and a mouse monoclonal antibody to β‐tubulin.

Techniques: Infection, Expressing

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Expression levels of ACE2, TMPRSS2, Kim‐1, and NRP‐1 proteins in different cell lines/types, and examination of ACE glycosylation in Huh‐7 cells analyzed by Western blot analysis. (A) Expression levels of ACE2, TMPRSS2, Kim‐1, and NRP‐1 proteins in different cell lines/types. Results showed that there are different expression profiles of ACE2, TMPRSS2, Kim‐1, and NRP‐1 in different cell lines/types. (B) Examination of ACE glycosylation in Huh‐7 cells. Untreated and PNGase F‐treated cell lysates were examined. β‐tubulin was used for loading normalization. Two bands of ~85 kD and ~120 kD were detected for ACE2 protein representing the unglycosylated and glycosylated forms of ACE2 (ungly‐ACE2 and gly‐ACE2), respectively. Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1; TMPRSS2, transmembrane serine protease 2

Article Snippet: The primary antibodies used were a rabbit polyclonal antibody to ACE2 (ab15348, Abcam), rabbit monoclonal antibodies to TMPRSS2 (EPR3861) (ab92323, Abcam), Kim‐1 (E1R9N) (14971 S, CST) and NRP‐1 (EPR3113) (ab81321, Abcam), and a mouse monoclonal antibody to β‐tubulin.

Techniques: Expressing, Western Blot

Journal: Journal of medical virology

Article Title: SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1 and NRP-1 in human cells from respiratory, urinary, digestive, reproductive and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Expression levels and patterns of ACE2, Kim‐1, and NRP‐1 proteins in nine selected cell types/lines analyzed by IFA. IFA, Immunofluorescence assay; Kim‐1, kidney injury molecule‐1; NRP‐1, neuropilin‐1

Article Snippet: The primary antibodies used were a rabbit polyclonal antibody to ACE2 (ab15348, Abcam), rabbit monoclonal antibodies to TMPRSS2 (EPR3861) (ab92323, Abcam), Kim‐1 (E1R9N) (14971 S, CST) and NRP‐1 (EPR3113) (ab81321, Abcam), and a mouse monoclonal antibody to β‐tubulin.

Techniques: Expressing, Immunofluorescence