p nephrin  (Santa Cruz Biotechnology)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 8. HQGZWWD increased the expression of Nephrin, Podocin in IgAN rats. (A) Nephrin gene expression in the renal tissue. (B) Nephrin immunohistochemical staining (magnification 400x). (C) Nephrin and podocin protein expression in the renal tissue. The numbers in the Western blotting figures represent groups: 1 means Control, 2 means Model, 3 means HQGZWWD low dose, 4 means HQGZWWD medium dose, 5 means HQGZWWD high dose, 6 means Valsartan. Data are expressed as the mean ± SD (N = 3). *P < 0.05,**P < 0.01 compared with respective control.

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Western Blot, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 11. HQGZWWD affected the distribution and expression of F-actin IgAN rats. (A) Double immunofluorescence of nephrin and F-actin (magnification 400x). (B) Double immunofluorescence of nephrin and F-actin (magnification 1000x).

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 15. HQGZWWD affected the distribution and expression of TNFR1 IgAN rats. (A) Double immunofluorescence of nephrin and TNFR1 (magnification 400x). (B) Double immunofluorescence of nephrin and TNFR1 (magnification 1000x).

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 14. HQGZWWD affected the expression of p-Nephrin and AT1R in IgAN rats. (A) AT1R gene expression in the renal tissue. (B) p-Nephrin and AT1R protein expression in the renal tissue. (C) AT1R immunohistochemical staining (magnification 400x). The numbers in the Western blotting figures represent groups: 1 means Control, 2 means Model, 3 means HQGZWWD low dose, 4 means HQGZWWD medium dose, 5 means HQGZWWD high dose, 6 means Valsartan. Data are expressed as the mean ± SD (N = 3). *P < 0.05,**P < 0.01 compared with respective control.

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Western Blot, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 17. HQGZWWD affected the distribution and expression of c-Abl IgAN rats. (A) Double immunofluorescence of nephrin and c-Abl (magnification 400x). (B) Double immunofluorescence of nephrin and c-Abl (magnification 1000x).

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huangqi Guizhi Wuwu Decoction attenuates Podocyte cytoskeletal protein damage in IgA nephropathy rats by regulating AT1R/Nephrin/c-Abl pathway.

doi: 10.1016/j.biopha.2021.111907

Figure Lengend Snippet: Fig. 16. HQGZWWD affected the distribution and expression of AT1R IgAN rats. (A) Double immunofluorescence of nephrin and AT1R (magnification 400x). (B) Double immunofluorescence of nephrin and AT1R actin (magnification 1000x).

Article Snippet: The membranes were blocked with 5% skimmed milk for 2 h, and incubated overnight at 4 ◦C with specific primary antibodies directed against podocin (P0372, Sigma), nephrin (ab216341, Abcam), p-nephrin (ab80298, Abcam), AT1R (ab124505, Abcam), TNFR1 (ab19139), α-ACTN4 (ab108198, Abcam), TNF-α (ab6671, Abcam), and c-ABL (2862, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten induces dephosphorylation of nephrin in a PTPase-dependent manner. Expression level of C1-Ten in ( a,b ) the kidney of db/db mice or ( c ) human podocyte cell line under high glucose (HG) condition. ( a ) Expression levels of C1-Ten were measured by Western blotting in whole kidney lysates of db/m and db/db mice. Data are means ± SEM (n = 3 mice per group). ( b ) Localization of endogenous C1-Ten was observed in the kidneys of db/m and db/db mice. Podocyte colocalization was revealed by immunofluorescence of C1-Ten (green), synaptopodin (red) and Hoechst (blue). Original magnification: X400 (scale bar, 50 µm) ( c ) Fully differentiated human podocytes were serum-starved for 18 h, then incubated in medium with normal glucose (NG) or HG for 24 h. Level of C1-Ten protein was measured. Data are means ± SEM (n = 3). ( d,e ) Effect of C1-Ten depletion on the HG-mediated reduction of nephrin phosphorylation. ( d ) C1-Ten knockdown was performed in human podocytes, which were then transfected with 50 nM of control or TNS2 siRNA (siC1-Ten), then stimulated with NG or HG for 24 h. Expression levels of C1-Ten were measured by Western blotting in total cell lysates. ( e ) Remaining cells lysates were subjected to immunoprecipitation with anti-nephrin antibodies. Immunoprecipitates were analyzed by immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. Data are means ± SEM (n = 3). ( f ) Effect of C1-Ten on Fyn-induced nephrin phosphorylation. 293NPHS cells were cotransfected with WT-Fyn and either FLAG vector, FLAG C1-Ten WT or FLAG C1-Ten CS. GFP nephrin was immunoprecipitated from cell lysates and subjected to immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. Data are means ± SEM (n = 3). *P < 0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: De-Phosphorylation Assay, Expressing, Western Blot, Immunofluorescence, Incubation, Phospho-proteomics, Knockdown, Transfection, Control, Immunoprecipitation, Plasmid Preparation

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: Nephrin competes with IRS-1, sequesters PI3K from IRS-1. ( a,b ) The preferential substrate of C1-Ten when IRS-1 and nephrin coexist. ( a ) FLAG C1-Ten CS was introduced with HA IRS-1 or GFP Nephrin into HEK293 cells, then immunoprecipitated with anti-FLAG beads and immunoblotted with IRS-1, GFP or FLAG. Data are means ± SEM (n = 3). ( b ) Nephrin knockdown was performed in human podocytes, which were transfected with 50 nM of control or NPHS1 siRNA (siNephrin). Cell lysates were subjected to immunoprecipitation with anti-IRS-1 antibodies. Immunoprecipitates were analyzed by immunoblotting with C1-Ten and IRS-1. Expression levels of nephrin, IRS-1 and actin were measured by Western blotting in total cell lysates. Data are means ± SEM (n = 3). ( c ) IRS-1 Y612 phosphorylation and PI3K interaction were monitored by increasing FLAG nephrin. HEK293 cells were transfected with FLAG nephrin. HEK293 cells were serum-starved for 18 h, then treated with 10 nM of insulin for 5 min, and subjected to immunoprecipitation with anti-IRS-1 antibody. The immunoprecipitates were subjected to immunoblotting with pY612 IRS-1, PI3K regulatory subunit α (p85α) or total IRS-1. Data are means ± SEM (n = 3). ( d ) Effect of nephrin on the insulin signaling. 293VEC or 293NPHS cells were serum-starved for 18 h, then treated with 10 nM insulin. Data are means ± SEM (n = 3). *P <0.05; **P <0.01.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Knockdown, Transfection, Control, Western Blot, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten PTPase activates mTORC1. ( a ) Effect of nephrin Y1138F mutant on IRS-1-mediated mTORC1 activation. HEK293 cells were transfected with GFP vector, GFP nephrin WT, or GFP nephrin Y1138F for 24 h. The cells were serum-starved for 18 h, then treated with 10 nM insulin for 30 min. mTORC1 activation was measured by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). ( b ) Effect of C1-Ten overexpression on mTORC1 activation. Human podocytes were transduced with Adenovirus (Ad) GFP, Ad C1-Ten WT or Ad C1-Ten CS for 48 h. Protein expression of phospho- and total-S6K1 were detected by Western blotting. Data are means ± SEM (n = 3). ( c ) Effect of C1-Ten knockdown on the HG-mediated mTORC1 activation. mTORC1 activation was presented by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). *P <0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Over Expression, Transduction, Expressing, Knockdown

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten PTPase causes kidney dysfunction in vivo . ( a ) GFP expression measured using Western blot analysis of whole kidney lysates. ( b ) Localization of adenovirus-mediated transgene expression. Mice were infected with adenovirus, then sacrificed 7 d later and their kidneys stained with GFP, phospho S6 (red) and Hoechst (blue). Original magnification: X400 (scale bar, 50 µm) ( c ) Effect of C1-Ten overexpression on the nephrin dephosphorylation and mTORC1 activation were measured in adenovirus-infected-whole kidney lysates. Immunoprecipitation was performed with anti-nephrin antibody, and the immunoprecipitates were analyzed by immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. mTORC1 activation was also measured by immunoblotting of phospho- and total- S6K1. ( d and e ) Morphological changes of glomeruli in adenovirus-infected kidneys. ( d ) Adenovirus-infected kidneys stained with periodic acid Schiff (PAS) reagent for renal pathology. Upper image: magnification X100 (scale bar, 100 µm); lower image: magnification X400 (scale bar, 20 µm). ( e ) After PAS staining, glomerular volume was analyzed using Meta Morph image analysis (20 glomeruli per animal). ( f ) Adenovirus-infected kidney podocytes were analyzed by transmission electron microscopy (TEM). TEM analysis revealed effaced podocyte foot process in the kidneys treated with C1-Ten WT (asterisks). Original magnification: X30000 (scale bar, 1 µm) ( g ) Urinary albumin excretion for 24 h was measured using ELISA. Data are means ± SEM (n = 3–5 mice per group). *P <0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vivo, Expressing, Western Blot, Infection, Staining, Over Expression, De-Phosphorylation Assay, Activation Assay, Immunoprecipitation, Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: Graphical summary. The balance of mTORC1 activity in normal podocytes is maintained by competition between nephrin and IRS-1 to PI3K. Under high glucose condition, upregulated C1-Ten acts as a PTPase at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. By activating mTORC1, excessive C1-Ten contributes to development of podocyte dysfunctions such as hypertrophy and proteinuria.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Binding Assay