nbp2 75624  (Novus Biologicals)

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Activation of TLR4/MyD88/PKCδ/SHP-1 signaling cascade was involved in hyperglycemic podocyte damage in vivo and in vitro . (A) The protein levels of TLR4 were determined in renal tissues from mice at 12 and 16 weeks by western blot analysis. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05 ( n = 8). (B) Representative western blot of the expression of MyD88, p-PKCδ (Y311), PKCδ and SHP-1 in renal cortex from mice at 12 and 16 weeks. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 8). (C) Representative double IF staining of glomerular SHP-1 and synaptopodin expression in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (D) The expression of TLR4, MyD88, p-PKCδ, PKCδ and SHP-1 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (E) IF staining of podocyte proteins p-cadherin and synaptopodin after LG or HG stimulation for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . ( n = 3 independent experiments). (F) Determination of SD protein podocin and injury marker desmin in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Activation Assay, In Vivo, In Vitro, Western Blot, Expressing, Staining, Cell Culture, Transfection, Marker

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Hyperglycemia triggered PKCδ activation and subsequent binding to downstream SHP-1 following TLR4/MyD88 signaling to induce podocyte damage. (A) Determination of expression of p-PKCδ, PKCδ and SHP-1 in cultured podocytes from treatments of HG for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; **** P < 0.001; ## P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (B) The expression of podocin and desmin in podocytes treated with HG for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (C) The representative western blot to show the expression of TLR4 and MyD88 in podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Data are presented as mean ± SD. P values were determined by one-way ANOVA and data are presented as mean ± SD; P > 0.05 ( n = 3 independent experiments). (DF) Surface diagram of the docking model and their interfacing residues between MyD88 and PKCδ (D) (MyD88, purple; PKCδ, yellow; hydrogen bonds, dotted line), and between PKCδ and SHP-1 (F) (PKCδ, yellow; SHP-1, blue; hydrogen bonds, dotted line). (EG) CO-IP analysis of the interaction between MyD88 and PKCδ (E), and between PKCδ and SHP-1 (G) in podocytes under HG condition in vitro . ( n = 3 independent experiments).

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Activation Assay, Binding Assay, Expressing, Cell Culture, Transfection, Western Blot, Co-Immunoprecipitation Assay, In Vitro

Journal: Scientific Reports

Article Title: Autoantibodies against nephrin and podocin are associated with disease severity and steroid dependence in adult-onset nephrotic syndrome

doi: 10.1038/s41598-026-43612-7

Figure Lengend Snippet: Serum anti-nephrin and anti-podocin antibody levels among glomerular disease groups. Scatter plots show the distribution of anti-nephrin (left) and anti-podocin (right) antibody levels in patients with MCNS, FSGS, PLA2R-MN, NELL1-MN, and controls. Horizontal dashed red lines indicate the ELISA cutoff values (0.2 AU/mL for anti-nephrin and 90 AU/mL for anti-podocin), determined as the upper limit of the control group. Patients double-positive for both antibodies are indicated by open circles of the same color. Anti-nephrin antibodies were predominantly detected in MCNS and FSGS, whereas anti-podocin antibodies were more frequently observed in NELL1-MN.

Article Snippet: Recombinant human Nephrin (Sino Biological, #17,757-H08H) and Podocin (R&D Systems, #9287-PO) were coated onto ELISA plates at 100 ng/well in coating buffer (BioLegend, #421,701) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Control