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Santa Cruz Biotechnology mouse anti nephrin monoclonal antibody

High-dose glucose-induced <t>nephrin</t> endocytosis promotes podocyte injury and endocytosis inhibitors block this effect. A and B: Western blot analysis of cytoplasmic nephrin, membrane nephrin, and total nephrin in podocytes after different treatments and densitometric quantification of these results ( n = 3); C: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue: Nuclei; red: Cytoskeleton); D: Adhesion of podocytes after different treatments ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 400; scale bar: 20 μm) and quantification of these results ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Mouse Anti Nephrin Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes"

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

Journal: World Journal of Diabetes

doi: 10.4239/wjd.v16.i10.109782

High-dose glucose-induced nephrin endocytosis promotes podocyte injury and endocytosis inhibitors block this effect. A and B: Western blot analysis of cytoplasmic nephrin, membrane nephrin, and total nephrin in podocytes after different treatments and densitometric quantification of these results ( n = 3); C: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue: Nuclei; red: Cytoskeleton); D: Adhesion of podocytes after different treatments ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 400; scale bar: 20 μm) and quantification of these results ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Figure Legend Snippet: High-dose glucose-induced nephrin endocytosis promotes podocyte injury and endocytosis inhibitors block this effect. A and B: Western blot analysis of cytoplasmic nephrin, membrane nephrin, and total nephrin in podocytes after different treatments and densitometric quantification of these results ( n = 3); C: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue: Nuclei; red: Cytoskeleton); D: Adhesion of podocytes after different treatments ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 400; scale bar: 20 μm) and quantification of these results ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Techniques Used: Blocking Assay, Western Blot, Membrane, Confocal Microscopy, Electron Microscopy

Rab5 promotes podocyte injury by increasing nephrin endocytosis, Rab5 silencing blocks this effect, and Rab5 upregulation promotes this effect. A and B: Western blot analysis of Rab5 in podocytes after different treatments and densitometric quantification of these results ( n = 3); C and D: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) in podocytes after different treatments and densitometric quantification of the C-nephrin/T-nephrin ratio ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 1000; scale bar: 50 μm) and quantification of these results ( n = 3); G: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue nuclei; red: Cytoskeleton); H: Adhesion of podocytes after different treatments ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NC: Negative control.

Figure Legend Snippet: Rab5 promotes podocyte injury by increasing nephrin endocytosis, Rab5 silencing blocks this effect, and Rab5 upregulation promotes this effect. A and B: Western blot analysis of Rab5 in podocytes after different treatments and densitometric quantification of these results ( n = 3); C and D: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) in podocytes after different treatments and densitometric quantification of the C-nephrin/T-nephrin ratio ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 1000; scale bar: 50 μm) and quantification of these results ( n = 3); G: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue nuclei; red: Cytoskeleton); H: Adhesion of podocytes after different treatments ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NC: Negative control.

Techniques Used: Western Blot, Electron Microscopy, Confocal Microscopy, Negative Control

Rab5-mediated nephrin endocytosis by podocytes depends on Rab5 activation. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes after different treatments and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes after different treatments (magnification: × 630; Scale bar: 20 μm); E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes after different treatments and quantification of these results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin and total nephrin in podocytes after different treatments and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Figure Legend Snippet: Rab5-mediated nephrin endocytosis by podocytes depends on Rab5 activation. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes after different treatments and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes after different treatments (magnification: × 630; Scale bar: 20 μm); E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes after different treatments and quantification of these results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin and total nephrin in podocytes after different treatments and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot

IL-6/Rab5 signaling mediates nephrin endocytosis in podocytes by promoting crosstalk between HK-2 cells and podocytes. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes cultured in different conditioned media and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes grown in different conditioned media (magnification: × 630; scale bar: 20 μm); E: Co-immunoprecipitation of nephrin and active Rab5 in podocytes grown in different conditioned media and quantification of these results ( n = 3; F: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) after growth in different conditioned media and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NAb: Neutralizing Ab.

Figure Legend Snippet: IL-6/Rab5 signaling mediates nephrin endocytosis in podocytes by promoting crosstalk between HK-2 cells and podocytes. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes cultured in different conditioned media and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes grown in different conditioned media (magnification: × 630; scale bar: 20 μm); E: Co-immunoprecipitation of nephrin and active Rab5 in podocytes grown in different conditioned media and quantification of these results ( n = 3; F: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) after growth in different conditioned media and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NAb: Neutralizing Ab.

Techniques Used: Immunoprecipitation, Cell Culture, Western Blot

Nicotinamide mononucleotide alleviates podocyte injury in diabetic mice. A and B: Blood glucose and HbA1c levels in the different groups; C: Systolic and diastolic blood pressure in the different groups; D and E: Representative PAS staining (magnification × 400; scale bar: 50 μm) and quantification of the glomerular surface area in different groups ( n = 10 glomeruli/group); F: Representative electron microscopy images of podocyte foot process effacement in the different groups (magnification × 10,000; scale bar: 100 μm); G: Urinary albumin excretion (albumin/creatinine ratio) in the different groups ( n = 5); H and I: Co-localization images and quantification of the epithelial-to-mesenchymal transition, indicated by altered E-cadherin and α-SMA expression, in the different groups (magnification × 630; scale bar: 20 μm); J and K: Immunohistochemical staining and quantification of glomerular expression of Rab5-GTP in the different groups (magnification × 400; scale bar: 20 μm); L and M: Immunofluorescence co-localization and quantification of nephrin and active Rab5 (Rab5-GTP) in the different groups (magnification × 630; scale bar: 20 μm). Data are expressed as mean ± SEM. a P < 0.05; b P < 0.001. DN: Diabetic nephropathy; NMN: Nicotinamide mononucleotide; AOC: Area of coverage.

Figure Legend Snippet: Nicotinamide mononucleotide alleviates podocyte injury in diabetic mice. A and B: Blood glucose and HbA1c levels in the different groups; C: Systolic and diastolic blood pressure in the different groups; D and E: Representative PAS staining (magnification × 400; scale bar: 50 μm) and quantification of the glomerular surface area in different groups ( n = 10 glomeruli/group); F: Representative electron microscopy images of podocyte foot process effacement in the different groups (magnification × 10,000; scale bar: 100 μm); G: Urinary albumin excretion (albumin/creatinine ratio) in the different groups ( n = 5); H and I: Co-localization images and quantification of the epithelial-to-mesenchymal transition, indicated by altered E-cadherin and α-SMA expression, in the different groups (magnification × 630; scale bar: 20 μm); J and K: Immunohistochemical staining and quantification of glomerular expression of Rab5-GTP in the different groups (magnification × 400; scale bar: 20 μm); L and M: Immunofluorescence co-localization and quantification of nephrin and active Rab5 (Rab5-GTP) in the different groups (magnification × 630; scale bar: 20 μm). Data are expressed as mean ± SEM. a P < 0.05; b P < 0.001. DN: Diabetic nephropathy; NMN: Nicotinamide mononucleotide; AOC: Area of coverage.

Techniques Used: Staining, Electron Microscopy, Expressing, Immunohistochemical staining, Immunofluorescence

Nicotinamide mononucleotide protection depends on crosstalk between proximal tubular epithelial cell and podocytes. A and B: Western blot analysis of epithelial-to-mesenchymal transition markers in HK-2 cells that received different treatments and quantification of these results ( n = 3); C: IL-6 secretion by HK-2 cells that received different treatments ( n = 3); D: Co-immunoprecipitation of active Rab5 in podocytes cultured in different conditioned media and quantification of these results; E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes cultured in different conditioned media and quantification of the results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) from podocytes cultured in different conditioned media and quantification of C-nephrin/T-nephrin ratio ( n = 3); I: Representative confocal microscopy images of podocytes cultured in different conditioned media (magnification: × 1000; scale bar: 50 μm; red: Cytoskeleton; blue: Nuclei); J: Quantification of podocyte adhesion after culture in different conditioned media ( n = 3); K: Representative electron microscopy images of podocyte spreading after culture in different conditioned media (magnification: × 400; scale bar: 20 μm); L: Quantitative analysis of podocyte spreading after culture in different conditioned media ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NMN: Nicotinamide mononucleotide.

Figure Legend Snippet: Nicotinamide mononucleotide protection depends on crosstalk between proximal tubular epithelial cell and podocytes. A and B: Western blot analysis of epithelial-to-mesenchymal transition markers in HK-2 cells that received different treatments and quantification of these results ( n = 3); C: IL-6 secretion by HK-2 cells that received different treatments ( n = 3); D: Co-immunoprecipitation of active Rab5 in podocytes cultured in different conditioned media and quantification of these results; E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes cultured in different conditioned media and quantification of the results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) from podocytes cultured in different conditioned media and quantification of C-nephrin/T-nephrin ratio ( n = 3); I: Representative confocal microscopy images of podocytes cultured in different conditioned media (magnification: × 1000; scale bar: 50 μm; red: Cytoskeleton; blue: Nuclei); J: Quantification of podocyte adhesion after culture in different conditioned media ( n = 3); K: Representative electron microscopy images of podocyte spreading after culture in different conditioned media (magnification: × 400; scale bar: 20 μm); L: Quantitative analysis of podocyte spreading after culture in different conditioned media ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NMN: Nicotinamide mononucleotide.

Techniques Used: Western Blot, Immunoprecipitation, Cell Culture, Confocal Microscopy, Electron Microscopy

Possible mechanism of crosstalk between proximal tubular epithelial cells and podocytes and effect of nicotinamide mononucleotide during diabetic nephropathy. High-dose glucose induces the epithelial-mesenchymal transition and secretion of IL-6 by proximal tubular epithelial cells (PTECs). Podocytes sense this IL-6, and this leads to the binding of internalized nephrin with active Rab5, followed by disruptions of the cytoskeleton, podocyte adhesion, and podocyte spreading. nicotinamide mononucleotide blocks signaling from the PTECs, decreases the binding of nephrin with active Rab5, and ameliorates cellular damage. EMT: Epithelial-mesenchymal transition; NMN: Nicotinamide mononucleotide.

Figure Legend Snippet: Possible mechanism of crosstalk between proximal tubular epithelial cells and podocytes and effect of nicotinamide mononucleotide during diabetic nephropathy. High-dose glucose induces the epithelial-mesenchymal transition and secretion of IL-6 by proximal tubular epithelial cells (PTECs). Podocytes sense this IL-6, and this leads to the binding of internalized nephrin with active Rab5, followed by disruptions of the cytoskeleton, podocyte adhesion, and podocyte spreading. nicotinamide mononucleotide blocks signaling from the PTECs, decreases the binding of nephrin with active Rab5, and ameliorates cellular damage. EMT: Epithelial-mesenchymal transition; NMN: Nicotinamide mononucleotide.

Techniques Used: Binding Assay

Image Search Results

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: High-dose glucose-induced nephrin endocytosis promotes podocyte injury and endocytosis inhibitors block this effect. A and B: Western blot analysis of cytoplasmic nephrin, membrane nephrin, and total nephrin in podocytes after different treatments and densitometric quantification of these results ( n = 3); C: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue: Nuclei; red: Cytoskeleton); D: Adhesion of podocytes after different treatments ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 400; scale bar: 20 μm) and quantification of these results ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Article Snippet: Mouse anti-nephrin monoclonal antibody (mAb; sc-376522), mouse anti-β-actin mAb (sc-47778), and Rab5 siRNA (sc-36344) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Techniques: Blocking Assay, Western Blot, Membrane, Confocal Microscopy, Electron Microscopy

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: Rab5 promotes podocyte injury by increasing nephrin endocytosis, Rab5 silencing blocks this effect, and Rab5 upregulation promotes this effect. A and B: Western blot analysis of Rab5 in podocytes after different treatments and densitometric quantification of these results ( n = 3); C and D: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) in podocytes after different treatments and densitometric quantification of the C-nephrin/T-nephrin ratio ( n = 3); E and F: Representative electron microscopy images of podocyte spreading after different treatments (magnification: × 1000; scale bar: 50 μm) and quantification of these results ( n = 3); G: Representative confocal microscopy images of podocytes after different treatments (magnification: × 1000; scale bar: 50 μm; blue nuclei; red: Cytoskeleton); H: Adhesion of podocytes after different treatments ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NC: Negative control.

Techniques: Western Blot, Electron Microscopy, Confocal Microscopy, Negative Control

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: Rab5-mediated nephrin endocytosis by podocytes depends on Rab5 activation. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes after different treatments and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes after different treatments (magnification: × 630; Scale bar: 20 μm); E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes after different treatments and quantification of these results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin and total nephrin in podocytes after different treatments and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose.

Techniques: Activation Assay, Immunoprecipitation, Western Blot

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: IL-6/Rab5 signaling mediates nephrin endocytosis in podocytes by promoting crosstalk between HK-2 cells and podocytes. A and B: Co-immunoprecipitation of active Rab5 (Rab5-GTP) in podocytes cultured in different conditioned media and quantification of these results ( n = 3); C and D: Representative co-localization images and quantitative of nephrin (green) and active Rab5 (red) in podocytes grown in different conditioned media (magnification: × 630; scale bar: 20 μm); E: Co-immunoprecipitation of nephrin and active Rab5 in podocytes grown in different conditioned media and quantification of these results ( n = 3; F: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) after growth in different conditioned media and quantification of the C-nephrin/T-nephrin ratio ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NAb: Neutralizing Ab.

Techniques: Immunoprecipitation, Cell Culture, Western Blot

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: Nicotinamide mononucleotide alleviates podocyte injury in diabetic mice. A and B: Blood glucose and HbA1c levels in the different groups; C: Systolic and diastolic blood pressure in the different groups; D and E: Representative PAS staining (magnification × 400; scale bar: 50 μm) and quantification of the glomerular surface area in different groups ( n = 10 glomeruli/group); F: Representative electron microscopy images of podocyte foot process effacement in the different groups (magnification × 10,000; scale bar: 100 μm); G: Urinary albumin excretion (albumin/creatinine ratio) in the different groups ( n = 5); H and I: Co-localization images and quantification of the epithelial-to-mesenchymal transition, indicated by altered E-cadherin and α-SMA expression, in the different groups (magnification × 630; scale bar: 20 μm); J and K: Immunohistochemical staining and quantification of glomerular expression of Rab5-GTP in the different groups (magnification × 400; scale bar: 20 μm); L and M: Immunofluorescence co-localization and quantification of nephrin and active Rab5 (Rab5-GTP) in the different groups (magnification × 630; scale bar: 20 μm). Data are expressed as mean ± SEM. a P < 0.05; b P < 0.001. DN: Diabetic nephropathy; NMN: Nicotinamide mononucleotide; AOC: Area of coverage.

Techniques: Staining, Electron Microscopy, Expressing, Immunohistochemical staining, Immunofluorescence

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: Nicotinamide mononucleotide protection depends on crosstalk between proximal tubular epithelial cell and podocytes. A and B: Western blot analysis of epithelial-to-mesenchymal transition markers in HK-2 cells that received different treatments and quantification of these results ( n = 3); C: IL-6 secretion by HK-2 cells that received different treatments ( n = 3); D: Co-immunoprecipitation of active Rab5 in podocytes cultured in different conditioned media and quantification of these results; E and F: Co-immunoprecipitation of nephrin and active Rab5 in podocytes cultured in different conditioned media and quantification of the results ( n = 3); G and H: Western blot analysis of cytoplasmic nephrin (C-nephrin) and total nephrin (T-nephrin) from podocytes cultured in different conditioned media and quantification of C-nephrin/T-nephrin ratio ( n = 3); I: Representative confocal microscopy images of podocytes cultured in different conditioned media (magnification: × 1000; scale bar: 50 μm; red: Cytoskeleton; blue: Nuclei); J: Quantification of podocyte adhesion after culture in different conditioned media ( n = 3); K: Representative electron microscopy images of podocyte spreading after culture in different conditioned media (magnification: × 400; scale bar: 20 μm); L: Quantitative analysis of podocyte spreading after culture in different conditioned media ( n = 3). a P < 0.05; b P < 0.001. HG: High-dose glucose; NMN: Nicotinamide mononucleotide.

Techniques: Western Blot, Immunoprecipitation, Cell Culture, Confocal Microscopy, Electron Microscopy

Journal: World Journal of Diabetes

Article Title: Nicotinamide mononucleotide protects against diabetic nephropathy via IL-6/Rab5-mediated crosstalk between proximal tubular epithelial cells and podocytes

doi: 10.4239/wjd.v16.i10.109782

Figure Lengend Snippet: Possible mechanism of crosstalk between proximal tubular epithelial cells and podocytes and effect of nicotinamide mononucleotide during diabetic nephropathy. High-dose glucose induces the epithelial-mesenchymal transition and secretion of IL-6 by proximal tubular epithelial cells (PTECs). Podocytes sense this IL-6, and this leads to the binding of internalized nephrin with active Rab5, followed by disruptions of the cytoskeleton, podocyte adhesion, and podocyte spreading. nicotinamide mononucleotide blocks signaling from the PTECs, decreases the binding of nephrin with active Rab5, and ameliorates cellular damage. EMT: Epithelial-mesenchymal transition; NMN: Nicotinamide mononucleotide.

Techniques: Binding Assay

Generation of TRPC6 knock-out and its coiled-coil deletion podocytes by CRISPR-Cas9 . A , schematic view of the TRPC6 gene coded by 13 exons. The screening of mutation by PCR sequencing resulted in a flame-shift stop codon of the single base pair insertion in exon 3 and the five base pair deletion in exon 12 that generated C6K/O and C6 Δ CC, respectively. B and C , sequences in exon 3 or exon 12 of the murine TRPC6 gene were chosen as target sequences (highlighted in orange ) for Cas9 enzyme. For generation of C6K/O, sequencing analysis of exon 3 confirmed single nucleotide insertion at 66,172 (NCBI Gene ID:22068) which resulted in partial protein at 186 a.a. For generation of C6 Δ CC, sequencing analysis of exon 12 confirmed deletion of five nucleotides at 135,657 which resulted in entire deletion of C-terminal CC domain. D and E , representative currents recorded from HEK293 cells expressing mouse TRPC6 WT and Δ CC variants. F , summary of residual currents after 5 s and 60 s from the peak. The columns and bars represent the mean and SD, respectively. Results for WT and Δ CC were compared using Student’s t test. Individual data points indicate individual cells measured. G , summaries of the fast inactivation kinetics. The fitting equation was as follows, I inactivation ( t ) = A 1 exp(-t/τ fast ) + A 2 exp(-t/τ slow ) + C, where I is current, t is time (sec); A 1 and A 2 are initial quantities; τ fast and τ slow are time constants; C is an offset, respectively. The columns and bars represent the mean and SD, respectively. p values were calculated using Student’s t test. Individual data points indicate individual cells measured. H , immunofluorescence staining of wild-type MPC-5 (WT), C6 Δ CC, and C6K/O cells with antibodies against TRPC6 (shown as magenta pseudocolor ) and nephrin ( green ). C6K/O cells showed little or no staining with TRPC6 antibody but robust signal with nephrin antibody.

Journal: The Journal of Biological Chemistry

Article Title: Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants

doi: 10.1016/j.jbc.2025.110256

Figure Lengend Snippet: Generation of TRPC6 knock-out and its coiled-coil deletion podocytes by CRISPR-Cas9 . A , schematic view of the TRPC6 gene coded by 13 exons. The screening of mutation by PCR sequencing resulted in a flame-shift stop codon of the single base pair insertion in exon 3 and the five base pair deletion in exon 12 that generated C6K/O and C6 Δ CC, respectively. B and C , sequences in exon 3 or exon 12 of the murine TRPC6 gene were chosen as target sequences (highlighted in orange ) for Cas9 enzyme. For generation of C6K/O, sequencing analysis of exon 3 confirmed single nucleotide insertion at 66,172 (NCBI Gene ID:22068) which resulted in partial protein at 186 a.a. For generation of C6 Δ CC, sequencing analysis of exon 12 confirmed deletion of five nucleotides at 135,657 which resulted in entire deletion of C-terminal CC domain. D and E , representative currents recorded from HEK293 cells expressing mouse TRPC6 WT and Δ CC variants. F , summary of residual currents after 5 s and 60 s from the peak. The columns and bars represent the mean and SD, respectively. Results for WT and Δ CC were compared using Student’s t test. Individual data points indicate individual cells measured. G , summaries of the fast inactivation kinetics. The fitting equation was as follows, I inactivation ( t ) = A 1 exp(-t/τ fast ) + A 2 exp(-t/τ slow ) + C, where I is current, t is time (sec); A 1 and A 2 are initial quantities; τ fast and τ slow are time constants; C is an offset, respectively. The columns and bars represent the mean and SD, respectively. p values were calculated using Student’s t test. Individual data points indicate individual cells measured. H , immunofluorescence staining of wild-type MPC-5 (WT), C6 Δ CC, and C6K/O cells with antibodies against TRPC6 (shown as magenta pseudocolor ) and nephrin ( green ). C6K/O cells showed little or no staining with TRPC6 antibody but robust signal with nephrin antibody.

Article Snippet: The cells were blocked and permeabilized with 0.5% Triton X-100/5% bovine serum albumin for 30 min. Then rabbit polyclonal anti-TRPC6 antibody (1:50, Alomone) and mouse monoclonal anti-nephrin antibody (1:50, Santa Cruz Biothecnology, sc-377246), or mouse monoclonal anti-synaptopodin antibody (1:2, Progen, clone G1D4) were incubate for overnight.

Techniques: Knock-Out, CRISPR, Mutagenesis, Sequencing, Generated, Expressing, Immunofluorescence, Staining

Detection of circulating anti-nephrin antibody by dual fluorescent cell transfection (cell based assay) method. ( A ) The green fluorescent is found, while the red fluorescent cannot be found, indicating a negative result of anti-nephrin antibody (MCD1–). ( B, C ) Both the green fluorescent and red fluorescent can be found, indicating a positive result of anti-nephrin antibody. Panel (B) shows anti-nephrin antibody 1:10 positive (MCD2+) and (C) shows antibody 1:32 positive (MCD3+). Scale bar: 20 μm.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Detection of circulating anti-nephrin antibody by dual fluorescent cell transfection (cell based assay) method. ( A ) The green fluorescent is found, while the red fluorescent cannot be found, indicating a negative result of anti-nephrin antibody (MCD1–). ( B, C ) Both the green fluorescent and red fluorescent can be found, indicating a positive result of anti-nephrin antibody. Panel (B) shows anti-nephrin antibody 1:10 positive (MCD2+) and (C) shows antibody 1:32 positive (MCD3+). Scale bar: 20 μm.

Article Snippet: After that, the second type of primary monoclonal mouse anti-human nephrin (Proteintech; 66970–1-lg) (dilution ratio 1:2500) were added, followed by HRP-labeled secondary goat anti-mouse IgG (Servicebio; GB23301) (dilution ratio 1:500), TSA dye and DAPI.

Techniques: Transfection, Cell Based Assay

Prevalence of circulating anti-nephrin antibody in patients with glomerular diseases and in controls. Panel shows the prevalence of anti-nephrin antibody in the MCD, FSGS, MN, DN, IgAN and controls.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Prevalence of circulating anti-nephrin antibody in patients with glomerular diseases and in controls. Panel shows the prevalence of anti-nephrin antibody in the MCD, FSGS, MN, DN, IgAN and controls.

Techniques:

Analysis of the discriminatory power of circulating anti-nephrin antibody in MCD. The graph shows the ROC curve analysis of circulating anti-nephrin antibody in discriminating patients with MCD from other type glomerular diseases and healthy controls.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Analysis of the discriminatory power of circulating anti-nephrin antibody in MCD. The graph shows the ROC curve analysis of circulating anti-nephrin antibody in discriminating patients with MCD from other type glomerular diseases and healthy controls.

Techniques:

Comparison of the clinical data of MCD patients based on anti-nephrin antibody status.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Comparison of the clinical data of MCD patients based on anti-nephrin antibody status.

Techniques: Comparison

Comparison of the clinical data in anti-nephrin antibody–negative MCD patients and positive MCD patients. ( A ) Anti-nephrin antibody–positive group patients had significantly heavier proteinuria. ( B ) Anti-nephrin antibody–positive group patients had significantly lower serum albumin level. ( C – E ) Anti-nephrin antibody–positive group patients had significantly higher cholesterol, triglyceride and LDL-C levels. ( F ) Anti-nephrin antibody–positive group patients had significantly lower serum IgG levels. Data are presented as median (25%–75%). * P < .05.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Comparison of the clinical data in anti-nephrin antibody–negative MCD patients and positive MCD patients. ( A ) Anti-nephrin antibody–positive group patients had significantly heavier proteinuria. ( B ) Anti-nephrin antibody–positive group patients had significantly lower serum albumin level. ( C – E ) Anti-nephrin antibody–positive group patients had significantly higher cholesterol, triglyceride and LDL-C levels. ( F ) Anti-nephrin antibody–positive group patients had significantly lower serum IgG levels. Data are presented as median (25%–75%). * P < .05.

Techniques: Comparison

Correlation between anti-nephrin antibody and clinical data in MCD patients.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Correlation between anti-nephrin antibody and clinical data in MCD patients.

Techniques:

Follow-up of four MCD patients with positive anti-nephrin antibody.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Follow-up of four MCD patients with positive anti-nephrin antibody.

Techniques:

Follow-up of MCD patients with positive anti-nephrin antibody. Panel shows the disease course, treatment therapy, the 24-h urinary protein levels and the anti-nephrin antibody status of the four MCD patients with positive circulating anti-nephrin antibody at study inclusion.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: Follow-up of MCD patients with positive anti-nephrin antibody. Panel shows the disease course, treatment therapy, the 24-h urinary protein levels and the anti-nephrin antibody status of the four MCD patients with positive circulating anti-nephrin antibody at study inclusion.

Techniques:

IgG deposition colocalized with nephrin in anti-nephrin antibody–positive MCD biopsies. ( A ) Immunofluorescence double-label staining image of glomerulus in anti-nephrin antibody–negative MCD (MCD–) biopsy. ( B, C ) Immunofluorescence double-label staining images of glomeruli in anti-nephrin antibody–positive MCD (MCD+) biopsies. There is an overlap (yellow) of IgG with nephrin (white arrows). Scale bar: 50 μm.

Journal: Clinical Kidney Journal

Article Title: Anti-nephrin antibody: a potential biomarker of minimal change disease

doi: 10.1093/ckj/sfaf012

Figure Lengend Snippet: IgG deposition colocalized with nephrin in anti-nephrin antibody–positive MCD biopsies. ( A ) Immunofluorescence double-label staining image of glomerulus in anti-nephrin antibody–negative MCD (MCD–) biopsy. ( B, C ) Immunofluorescence double-label staining images of glomeruli in anti-nephrin antibody–positive MCD (MCD+) biopsies. There is an overlap (yellow) of IgG with nephrin (white arrows). Scale bar: 50 μm.

Techniques: Immunofluorescence, Staining

SGK3 was associated with proteinuria and nephrin expression in ADR nephritis. An intravenous injection of ADR (10.5 mg/kg) was used to induce nephritis mouse model. (A) BUN, serum creatinine, and the urinary albumin/creatinine ratio were measured in mice after treatment with ADR 28 days. (B) Morphologically, PAS staining show tubules were frequently dilated and contained some casts after ADR injection. (C) Top panel: immunoblot analysis for nephrin and SGK3, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. (D) Top panel: immunohistochemical staining shows SGK3 and nephrin expression in the glomeruli after ADR injury. Representative images from six mice in each group are shown. Bottom panel: computerized morphometric quantification of SGK3 and nephrin staining in the glomeruli of control and ADR mice. (E) Representative immunofluorescent images showed staining with specific antibodies to SGK3 (red) and nephrin (green) in the glomeruli of control and ADR mice. Arrowheads indicate the colocalization of SGK3 and nephrin. (F) There was a significantly negative correlation between SGK3 expression and proteinuria levels in BALB/c mice ( n = 5 in control group, n = 6 in ADR group). (G) There was a significantly positive correlation between SGK3 and nephrin expression in BALB/c mice ( n = 5 in control group, n = 7 in ADR group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: SGK3 was associated with proteinuria and nephrin expression in ADR nephritis. An intravenous injection of ADR (10.5 mg/kg) was used to induce nephritis mouse model. (A) BUN, serum creatinine, and the urinary albumin/creatinine ratio were measured in mice after treatment with ADR 28 days. (B) Morphologically, PAS staining show tubules were frequently dilated and contained some casts after ADR injection. (C) Top panel: immunoblot analysis for nephrin and SGK3, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. (D) Top panel: immunohistochemical staining shows SGK3 and nephrin expression in the glomeruli after ADR injury. Representative images from six mice in each group are shown. Bottom panel: computerized morphometric quantification of SGK3 and nephrin staining in the glomeruli of control and ADR mice. (E) Representative immunofluorescent images showed staining with specific antibodies to SGK3 (red) and nephrin (green) in the glomeruli of control and ADR mice. Arrowheads indicate the colocalization of SGK3 and nephrin. (F) There was a significantly negative correlation between SGK3 expression and proteinuria levels in BALB/c mice ( n = 5 in control group, n = 6 in ADR group). (G) There was a significantly positive correlation between SGK3 and nephrin expression in BALB/c mice ( n = 5 in control group, n = 7 in ADR group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group.

Article Snippet: Monoclonal mouse anti–phospho-GSK3(α/β) antibody for immunoblotting, Polyclonal rabbit anti-SGK3 antibody for immunofluorescence, and monoclonal mouse anti-nephrin antibody for ubiquitination and immunofluorescence were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Techniques: Expressing, Injection, Staining, Western Blot, Immunohistochemical staining, Control

SGK3 was involved in ADR-induced downregulation of nephrin. Mouse podocytes were transiently transfected with SGK3-K191M plasmid for 48 h. (A) Top panel: immunoblot analysis for nephrin and SGK3 expression in total protein, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. (B) Top panel: immunoblot analysis for nephrin in membrane protein, Na,K-ATPase was used for normalization. (C) MPCs were transiently infected with SGK3 -shRNA for 72 h. Left panel: immunoblot analysis for nephrin and SGK3 expression; GAPDH was used for normalization. Right panel: Quantification of nephrin and SGK3 by densitometry.* P < 0.05, ** P < 0.01 vs. control group. (D) MPCs were transiently transfected with SGK3-S486D plasmid for 48 h and ADR was applied at last 24 h. Left: immunoblot analysis for nephrin and SGK3 expression; GAPDH was used for normalization. Right: Quantification of nephrin and SGK3 by densitometry. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. ADR-/S486D- group, # P < 0.05, ### P < 0.001 vs. ADR + /S486D- group.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: SGK3 was involved in ADR-induced downregulation of nephrin. Mouse podocytes were transiently transfected with SGK3-K191M plasmid for 48 h. (A) Top panel: immunoblot analysis for nephrin and SGK3 expression in total protein, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. (B) Top panel: immunoblot analysis for nephrin in membrane protein, Na,K-ATPase was used for normalization. (C) MPCs were transiently infected with SGK3 -shRNA for 72 h. Left panel: immunoblot analysis for nephrin and SGK3 expression; GAPDH was used for normalization. Right panel: Quantification of nephrin and SGK3 by densitometry.* P < 0.05, ** P < 0.01 vs. control group. (D) MPCs were transiently transfected with SGK3-S486D plasmid for 48 h and ADR was applied at last 24 h. Left: immunoblot analysis for nephrin and SGK3 expression; GAPDH was used for normalization. Right: Quantification of nephrin and SGK3 by densitometry. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. ADR-/S486D- group, # P < 0.05, ### P < 0.001 vs. ADR + /S486D- group.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Membrane, Infection, shRNA, Control

ADR treatment leaded to increased ubiquitination degradation of nephrin. Mouse podocytes were treated with 0.2 μg/ml ADR for 24 h. (A) Top panel: immunoblot analysis for desmin and podocin, GAPDH was used for normalization. Bottom panel: Quantification of desmin and podocin by densitometry. (B) Top panel: immunoblot analysis for nephrin and SGK3, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. Data are means ± SE of three independent experiments. * P < 0.05, ** P < 0.01 vs. control group. (C) MPCs were treated with ADR (0.2 μg/ml) and then treated with or without MG132 for 8 h. Top panel: immunoblot analysis for nephrin expression. Bottom panel: Quantification of nephrin by densitometry. ** P < 0.01 vs. ADR-/MG132- group, ## P < 0.01 vs. ADR + /MG132- group. (D) MPCs were transiently transfected with ubiquitin-Flag (UB-Flag) plasmid for 48 h then ADR was added for 24 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were blotted with anti-ubiquitin and anti-nephrin antibodies.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: ADR treatment leaded to increased ubiquitination degradation of nephrin. Mouse podocytes were treated with 0.2 μg/ml ADR for 24 h. (A) Top panel: immunoblot analysis for desmin and podocin, GAPDH was used for normalization. Bottom panel: Quantification of desmin and podocin by densitometry. (B) Top panel: immunoblot analysis for nephrin and SGK3, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and SGK3 by densitometry. Data are means ± SE of three independent experiments. * P < 0.05, ** P < 0.01 vs. control group. (C) MPCs were treated with ADR (0.2 μg/ml) and then treated with or without MG132 for 8 h. Top panel: immunoblot analysis for nephrin expression. Bottom panel: Quantification of nephrin by densitometry. ** P < 0.01 vs. ADR-/MG132- group, ## P < 0.01 vs. ADR + /MG132- group. (D) MPCs were transiently transfected with ubiquitin-Flag (UB-Flag) plasmid for 48 h then ADR was added for 24 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were blotted with anti-ubiquitin and anti-nephrin antibodies.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Expressing, Transfection, Plasmid Preparation

SGK3 inactivation leaded to increase ubiquitination degradation of nephrin. (A) MPCs were transiently transfected with SGK3-K191M plasmid for 48 h and MG132 was applied at last 8 h. Left: immunoblot analysis and quantification for SGK3 expression; GAPDH was used for normalization. Right: immunoblot analysis and quantification for nephrin expression; GAPDH was used for normalization. * P < 0.05, ** P < 0.01 vs. K191M-/MG132- group, # P < 0.05 vs. K191M + /MG132- group. (B) MPCs were transiently co-transfected with SGK3-K191M and ubiquitin-Flag (UB-Flag) plasmids for 48 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-Flag antibody and the immunoprecipitates were immunoblotted with anti-nephrin and anti-Flag antibodies. The protein expression levels of SGK3 were measured in the WCLs with immunoblot assays.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: SGK3 inactivation leaded to increase ubiquitination degradation of nephrin. (A) MPCs were transiently transfected with SGK3-K191M plasmid for 48 h and MG132 was applied at last 8 h. Left: immunoblot analysis and quantification for SGK3 expression; GAPDH was used for normalization. Right: immunoblot analysis and quantification for nephrin expression; GAPDH was used for normalization. * P < 0.05, ** P < 0.01 vs. K191M-/MG132- group, # P < 0.05 vs. K191M + /MG132- group. (B) MPCs were transiently co-transfected with SGK3-K191M and ubiquitin-Flag (UB-Flag) plasmids for 48 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-Flag antibody and the immunoprecipitates were immunoblotted with anti-nephrin and anti-Flag antibodies. The protein expression levels of SGK3 were measured in the WCLs with immunoblot assays.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Expressing

SGK3/Nedd4-2 signaling pathway regulated protein expression of nephrin through the ubiquitin-proteasome pathway. (A) MPCs were treated with 0.2 μg/ml ADR for 24 h. Top panel: immunoblot analysis for phospho-Nedd4-2 and Nedd4-2 expression. Bottom panel: Quantification of the ratio of phospho-Nedd4-2 to Nedd4-2 by densitometry. (B) MPCs were transiently transfected with SGK3-K191M plasmid for 48 h. Top panel: immunoblot analysis for phospho-Nedd4-2 and Nedd4-2 expression. Bottom panel: Quantification of the ratio of phospho-Nedd4-2 to Nedd4-2 by densitometry. (C) MPCs were infected with scramble (control) or Nedd4-2 -specific shRNAs for 72 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry. (D,E) MPCs were transiently transfected with Nedd4-2 WT plasmid for 48 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry (D). Top panel: immunoblot analysis for nephrin in membrane protein, Na,K-ATPase was used for normalization (E). Bottom panel: Quantification of nephrin by densitometry. * P < 0.05, ** P < 0.01 vs. control group. (F) MPCs were transiently transfected with Nedd4-2 WT plasmid for 48 h and MG132 was applied at last 8 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry. * P < 0.05 vs. Nedd4-2-/MG132- group, # P < 0.05 vs. Nedd4-2 + /MG132- group. (G) Network view of predicted E3-substrate interactions in UbiBrowser web services. In network view, the central node is NPHS1 (nephrin), as a substrate, and the surrounding nodes are the predicted E3 ligases. The width of the edge reflects the confidence of the interaction. (H) The possible binding regions of NPHS1 (nephrin) and Nedd4-2. (I) Mouse podocytes were transiently transfected with Nedd4-2 WT plasmids for 48 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were blotted with anti-Nedd4-2, anti-ubiquitin and anti-nephrin antibodies. The protein expression levels of Nedd4-2 in WCLs were analyzed with an immunoblot. (J) Representative immunofluorescent images showed staining with specific antibodies to Nedd4-2 (green) and nephrin (red) in the glomeruli of control and ADR mice. Arrowheads indicate the colocalization of Nedd4-2 and nephrin.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: SGK3/Nedd4-2 signaling pathway regulated protein expression of nephrin through the ubiquitin-proteasome pathway. (A) MPCs were treated with 0.2 μg/ml ADR for 24 h. Top panel: immunoblot analysis for phospho-Nedd4-2 and Nedd4-2 expression. Bottom panel: Quantification of the ratio of phospho-Nedd4-2 to Nedd4-2 by densitometry. (B) MPCs were transiently transfected with SGK3-K191M plasmid for 48 h. Top panel: immunoblot analysis for phospho-Nedd4-2 and Nedd4-2 expression. Bottom panel: Quantification of the ratio of phospho-Nedd4-2 to Nedd4-2 by densitometry. (C) MPCs were infected with scramble (control) or Nedd4-2 -specific shRNAs for 72 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry. (D,E) MPCs were transiently transfected with Nedd4-2 WT plasmid for 48 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry (D). Top panel: immunoblot analysis for nephrin in membrane protein, Na,K-ATPase was used for normalization (E). Bottom panel: Quantification of nephrin by densitometry. * P < 0.05, ** P < 0.01 vs. control group. (F) MPCs were transiently transfected with Nedd4-2 WT plasmid for 48 h and MG132 was applied at last 8 h. Top panel: immunoblot analysis for nephrin and Nedd4-2 expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and Nedd4-2 by densitometry. * P < 0.05 vs. Nedd4-2-/MG132- group, # P < 0.05 vs. Nedd4-2 + /MG132- group. (G) Network view of predicted E3-substrate interactions in UbiBrowser web services. In network view, the central node is NPHS1 (nephrin), as a substrate, and the surrounding nodes are the predicted E3 ligases. The width of the edge reflects the confidence of the interaction. (H) The possible binding regions of NPHS1 (nephrin) and Nedd4-2. (I) Mouse podocytes were transiently transfected with Nedd4-2 WT plasmids for 48 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were blotted with anti-Nedd4-2, anti-ubiquitin and anti-nephrin antibodies. The protein expression levels of Nedd4-2 in WCLs were analyzed with an immunoblot. (J) Representative immunofluorescent images showed staining with specific antibodies to Nedd4-2 (green) and nephrin (red) in the glomeruli of control and ADR mice. Arrowheads indicate the colocalization of Nedd4-2 and nephrin.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Infection, Control, Membrane, Binding Assay, Staining

SGK3/GSK3β signaling pathway regulated protein expression of nephrin. (A) Mouse podocytes were transiently transfected with SGK3-K191M plasmid for 48 h. Top panel: immunoblot analysis for phospho-GSK3β and GSK3β expression. Bottom panel: Quantification of the ratio of phospho-GSK3β to GSK3β by densitometry. (B) MPCs were transiently transfected with GSK3 β plasmid for 48 h. Top panel: immunoblot analysis for nephrin and GSK3β expression, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and GSK3β by densitometry. (C) MPCs were treated with 10 μM LiCl for 24 h. Left panel: immunoblot analysis of phospho-GSK3β and GSK3β and nephrin expression. Right panel: Quantification of nephrin by densitometry. * P < 0.05, ** P < 0.01 vs. control group. (D) MPCs were transiently transfected with GSK3 β plasmid for 48 h and MG132 was applied at last 8 h. Top panel: immunoblot analysis for nephrin and GSK3β expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and GSK3β by densitometry. * P < 0.05 vs. GSK3β-/MG132- group. (E) Mouse podocytes were transiently co-transfected with GSK3 β and ubiquitin-Flag (UB-Flag) plasmids for 48 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were immunoblotted with anti-ubiquitin and anti-nephrin antibodies. The protein expression levels of GSK3β in WCLs were analyzed by immunoblot.

Journal: Frontiers in Physiology

Article Title: Serum and Glucocorticoid-Inducible Kinase 3/Nedd4-2 Signaling Pathway Participates in Podocyte Injury by Regulating the Stability of Nephrin

doi: 10.3389/fphys.2021.810473

Figure Lengend Snippet: SGK3/GSK3β signaling pathway regulated protein expression of nephrin. (A) Mouse podocytes were transiently transfected with SGK3-K191M plasmid for 48 h. Top panel: immunoblot analysis for phospho-GSK3β and GSK3β expression. Bottom panel: Quantification of the ratio of phospho-GSK3β to GSK3β by densitometry. (B) MPCs were transiently transfected with GSK3 β plasmid for 48 h. Top panel: immunoblot analysis for nephrin and GSK3β expression, GAPDH was used for normalization. Bottom panel: Quantification of nephrin and GSK3β by densitometry. (C) MPCs were treated with 10 μM LiCl for 24 h. Left panel: immunoblot analysis of phospho-GSK3β and GSK3β and nephrin expression. Right panel: Quantification of nephrin by densitometry. * P < 0.05, ** P < 0.01 vs. control group. (D) MPCs were transiently transfected with GSK3 β plasmid for 48 h and MG132 was applied at last 8 h. Top panel: immunoblot analysis for nephrin and GSK3β expression; GAPDH was used for normalization. Bottom panel: Quantification of nephrin and GSK3β by densitometry. * P < 0.05 vs. GSK3β-/MG132- group. (E) Mouse podocytes were transiently co-transfected with GSK3 β and ubiquitin-Flag (UB-Flag) plasmids for 48 h and MG132 was applied at last 8 h. Whole-cell lysates were precipitated with an anti-nephrin antibody and the immunoprecipitates were immunoblotted with anti-ubiquitin and anti-nephrin antibodies. The protein expression levels of GSK3β in WCLs were analyzed by immunoblot.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Ubiquitin Proteomics

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