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Bioss anti myd88
Anti Myd88, supplied by Bioss, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+myd88/MyD88+Polyclonal+Antibody/pmc13058937-101-14-17
Average 94 stars, based on 59 article reviews

anti myd88 - by Bioz Stars, 2026-09

94/100 stars

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Incubation:

Article Title: Resveratrol affects the expression of uric acid transporter by improving inflammation.
Article Snippet: .. Lysates containing 50 μg protein were separated by SDS‐PAGE (stacking gel, 10%; resolving gel, 5%) and wet electro‐transferred to nitrocellulose membranes, which were blocked in buffer containing 5% skimmed milk powder at room temperature for 4 h. The membranes were then incubated with the following primary antibodies (dilution, 1:1,000) at 4 ̊C overnight: Anti‐GLUT9 (cat. no. 26486‐1‐AP), anti‐URAT1 (cat. no. 14937‐1‐AP), anti‐monocyte chemotactic protein‐1 (MCP‐1; cat no. 25542‐1‐AP), anti‐NLRP3 (cat. no. a5652; ABclonal Biotech Co., Ltd.), anti‐apoptosis‐associ‐ ated speck‐like protein (ASC; cat. no. 67494‐1‐Ig), anti‐MyD88 (cat. no. BS‐1047R; BIOSS), anti‐TNF receptor‐associated factor 6 (TRAF6; cat. no. 66498‐1‐Ig), anti‐TGF‐β‐activated kinase 1 (TAK1; cat. no. 12330‐2‐AP), anti‐NF‐κB p65 (cat. no. 66535‐1‐Ig), anti‐caspase‐1 (cat. no. 22915‐1‐AP), anti‐TLR4 (cat. no. 19811‐1‐AP) and anti‐β‐actin (cat. no. 20536‐1‐AP) (Remaining antibodies except anti‐NLRP3 and anti‐MyD8 all purchased from ProteinTech Group, Inc.). .. After three washes with Tris‐buffered saline‐Tween‐20 (20%) solution, the membranes were incubated with horseradish peroxidase‐conju‐ gated secondary antibodies (anti‐rabbit secondary antibody; dilution,1:8,000; cat. no. SA00001‐2; ProteinTech Group, Inc.; anti‐mouse secondary antibodies; dilution, 1:8,000; cat. no. GB23301; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature.

Article Title: Resveratrol affects the expression of uric acid transporter by improving inflammation
Article Snippet: .. Lysates containing 50 μg protein were separated by SDS-PAGE (stacking gel, 10%; resolving gel, 5%) and wet electro-transferred to nitrocellulose membranes, which were blocked in buffer containing 5% skimmed milk powder at room temperature for 4 h. The membranes were then incubated with the following primary antibodies (dilution, 1:1,000) at 4°C overnight: Anti-GLUT9 (cat. no. 26486-1-AP), anti-URAT1 (cat. no. 14937-1-AP), anti-monocyte chemotactic protein-1 (MCP-1; cat no. 25542-1-AP), anti-NLRP3 (cat. no. a5652; ABclonal Biotech Co., Ltd.), anti-apoptosis-associated speck-like protein (ASC; cat. no. 67494-1-Ig), anti-MyD88 (cat. no. BS-1047R; BIOSS), anti-TNF receptor-associated factor 6 (TRAF6; cat. no. 66498-1-Ig), anti-TGF-β-activated kinase 1 (TAK1; cat. no. 12330-2-AP), anti-NF-κB p65 (cat. no. 66535-1-Ig), anti-caspase-1 (cat. no. 22915-1-AP), anti-TLR4 (cat. no. 19811-1-AP) and anti-β-actin (cat. no. 20536-1-AP) (Remaining antibodies except anti-NLRP3 and anti-MyD8 all purchased from ProteinTech Group, Inc.). .. After three washes with Tris-buffered saline-Tween-20 (20%) solution, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit secondary antibody; dilution,1:8,000; cat. no. SA00001-2; ProteinTech Group, Inc.; anti-mouse secondary antibodies; dilution, 1:8,000; cat. no. GB23301; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature.

Article Title: Baicalin Rescues Cognitive Dysfunction, Mitigates Neurodegeneration, and Exerts Anti-Epileptic Effects Through Activating TLR4/MYD88/Caspase-3 Pathway in Rats
Article Snippet: .. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody antiCaspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). ..

Article Title: Baicalin Rescues Cognitive Dysfunction, Mitigates Neurodegeneration, and Exerts Anti-Epileptic Effects Through Activating TLR4/MYD88/Caspase-3 Pathway in Rats
Article Snippet: .. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody anti-Caspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs-1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). ..

SDS Page:

Expressing:

Article Title: Glycoside oleandrin downregulates toll-like receptor pathway genes and associated miRNAs in human melanoma cells.
Article Snippet: Melanoma accounts for the majority of skin cancer-related deaths.. Nerium oleander is a plant known to be toxic and consumed due to the cardiac glycosides it contains.. Oleandrin is a cardiac glycoside obtained from of N. oleander.

Western Blot:

Article Title: Panax notoginseng protects blood-retinal barrier by modulating microglia polarization via TLR4/MyD88/NF-κB pathway in endotoxin-induced uveitis.
Article Snippet: Background: Panax notoginseng is a traditional Chinese medicine for promoting blood circulation and removing blood stasis.. Panax notoginseng compound is widely used in the treatment of uveitis, however, the current research focuses on the compound, and the role of Panax notoginseng in the compound has not been elucidated.. Objective: The aim of this study was to explore the effects and potential molecular mechanism of Panax notoginseng against uveitis.

Blocking Assay:

Article Title: Baicalin Rescues Cognitive Dysfunction, Mitigates Neurodegeneration, and Exerts Anti-Epileptic Effects Through Activating TLR4/MYD88/Caspase-3 Pathway in Rats
Article Snippet: .. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody anti-Caspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs-1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). ..

Immunofluorescence:

Image Search Results

The TLR2-NF-κB signaling pathway plays a critical role in the radioprotective effect of Zymosan-A (A) Heatmap of differential gene expression between wild-type mice and Zymosan-A mice. (B) Scatterplot of differently expressed genes in ovary tissue after Zymosan-A treatment. Each dot stands for a gene. Red and green color dots indicate an increase or decrease, respectively. (C) Pathway enrichment analysis of KEGG pathways within the core network. (D) RNA level to verify the expression of DEGs, including TLR2, CCL3, CCL5, AKT1, MYD88, and IκBκB. (E) The expression of TLR2-NF-κB pathway-related proteins. (F) The serum E2 level, AMH level, FSH level, and LH level were measured at TLR2 KO + IR + NS group and TLR2 KO + IR + Zymosan-A group. (G) The cell viability in siTLR2 + NS + IR group and siTLR2 + Zymosan-A + IR group (error bars represent the mean ± SD of independent experiments; N.S., no statistical difference, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n = 3).

Journal: iScience

Article Title: Protective effect of Zymosan-A against radiation-induced premature ovarian insufficiency in a murine model

doi: 10.1016/j.isci.2026.115027

Figure Lengend Snippet: The TLR2-NF-κB signaling pathway plays a critical role in the radioprotective effect of Zymosan-A (A) Heatmap of differential gene expression between wild-type mice and Zymosan-A mice. (B) Scatterplot of differently expressed genes in ovary tissue after Zymosan-A treatment. Each dot stands for a gene. Red and green color dots indicate an increase or decrease, respectively. (C) Pathway enrichment analysis of KEGG pathways within the core network. (D) RNA level to verify the expression of DEGs, including TLR2, CCL3, CCL5, AKT1, MYD88, and IκBκB. (E) The expression of TLR2-NF-κB pathway-related proteins. (F) The serum E2 level, AMH level, FSH level, and LH level were measured at TLR2 KO + IR + NS group and TLR2 KO + IR + Zymosan-A group. (G) The cell viability in siTLR2 + NS + IR group and siTLR2 + Zymosan-A + IR group (error bars represent the mean ± SD of independent experiments; N.S., no statistical difference, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n = 3).

Article Snippet: The following antibodies were used: TLR2 (Cat no: 17236-1-AP, 1:1000, Proteintech), BCL-2 (Cat no: 60178-1-Ig, 1:1000, Proteintech), BAX (Cat no: 60267-1-Ig, 1:1000, Proteintech), PCNA (Cat no: 13110, 1:1000, CST), Cleaved Caspase-3 (Cat no: 9661, 1:1000, CST) and GAPDH (Cat no:5174, 1:1000, CST), p -IKKα/β (Cat no: 2697, 1:1000, CST), p-P65 (Cat no: 3033, 1:1000, CST), TLR2 (Cat no: 66645-1-Ig, 1:1000, Proteintech), Myd88 (Cat no: 50010, 1:1000, CST).

Techniques: Gene Expression, Expressing

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).

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

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).

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).

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

TLR4/MyD88/PKCδ/SHP-1 signaling cascade induced podocyte damage through regulating ER stress in DKD and podocyte damage. (A) The protein levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 were determined in renal tissues from mice at 12 and 16 weeks by western blot. 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). (B) Representative double IF staining of the expression ATF4 and podocyte marker synaptopodin in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (C) Representative western blot to assess the levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 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 ( n = 3 independent experiments). (D) ER morphological alterations and ER stress illustrated by ER-tracker staining in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or MCP-1 shRNAs, or pretreatment of 4-PBA. Scale bar, 10 μm. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; # P < 0.05 ( n = 3 independent experiments). (E) Representative western blot to detect the expression of ER stress proteins BIP, sXBP-1, ATF4 and ATF6 in cultured podocytes after HG stimulation 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 < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

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: TLR4/MyD88/PKCδ/SHP-1 signaling cascade induced podocyte damage through regulating ER stress in DKD and podocyte damage. (A) The protein levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 were determined in renal tissues from mice at 12 and 16 weeks by western blot. 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). (B) Representative double IF staining of the expression ATF4 and podocyte marker synaptopodin in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (C) Representative western blot to assess the levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 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 ( n = 3 independent experiments). (D) ER morphological alterations and ER stress illustrated by ER-tracker staining in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or MCP-1 shRNAs, or pretreatment of 4-PBA. Scale bar, 10 μm. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; # P < 0.05 ( n = 3 independent experiments). (E) Representative western blot to detect the expression of ER stress proteins BIP, sXBP-1, ATF4 and ATF6 in cultured podocytes after HG stimulation 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 < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Techniques: Western Blot, Staining, Expressing, Marker, Cell Culture, Transfection

TLR4/MyD88/PKCδ/SHP-1 signaling-dependent ER stress stimulated MCP-1 production via enhanced transcription activity by ATF4 binding to its promoter in hyperglycemic podocytes. (A) The altered expression of MCP-1 in renal tissue from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01 ( n = 8 samples). (B) Representative western blot to assess the levels of MCP-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 ( n = 3 independent experiments). (C) Representative western blot to show the expression of MCP-1 in podocytes after HG stimulation 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 < 0.01 ( n = 3 independent experiments). (DE) The expression of MCP-1 by western blot (D), and ER stress markers BIP, sXBP-1, ATF4 and ATF6 (E) after HG stimulation for 48 h with or without MCP-1 or scrambled shRNA transfection. 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). (F) Schematic description of primers spanning the distal, medial, and proximal regions of the MCP-1 promoter, encompassing predicted binding sites in the present study. (GH) ChIP analysis using anti-ATF4 or anti-IgG antibodies were performed either in cultured mouse podocytes after LG or HG treatment for 48 h (G), or HG stimulation with transfection of TLR4, PKCδ, MCP-1 or scrambled shRNA, or pretreatment of 4-PBA (H). Real-time PCR was used for the detection of the ChIP signals. P values were determined by Student’s t -test, or by one-way ANOVA, and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; **** P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (I) Determination of ATF4 nuclear accumulation in podocytes under LG or HG conditions by western blot. P values were determined by Student’s t -test and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (J) Assessment of nuclear ATF4 protein levels in cultured podocytes transfected with TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreated with 4-PBA under HG conditions for 48 h. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; * P < 0.01 ( n = 3 independent experiments).

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: TLR4/MyD88/PKCδ/SHP-1 signaling-dependent ER stress stimulated MCP-1 production via enhanced transcription activity by ATF4 binding to its promoter in hyperglycemic podocytes. (A) The altered expression of MCP-1 in renal tissue from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01 ( n = 8 samples). (B) Representative western blot to assess the levels of MCP-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 ( n = 3 independent experiments). (C) Representative western blot to show the expression of MCP-1 in podocytes after HG stimulation 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 < 0.01 ( n = 3 independent experiments). (DE) The expression of MCP-1 by western blot (D), and ER stress markers BIP, sXBP-1, ATF4 and ATF6 (E) after HG stimulation for 48 h with or without MCP-1 or scrambled shRNA transfection. 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). (F) Schematic description of primers spanning the distal, medial, and proximal regions of the MCP-1 promoter, encompassing predicted binding sites in the present study. (GH) ChIP analysis using anti-ATF4 or anti-IgG antibodies were performed either in cultured mouse podocytes after LG or HG treatment for 48 h (G), or HG stimulation with transfection of TLR4, PKCδ, MCP-1 or scrambled shRNA, or pretreatment of 4-PBA (H). Real-time PCR was used for the detection of the ChIP signals. P values were determined by Student’s t -test, or by one-way ANOVA, and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; **** P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (I) Determination of ATF4 nuclear accumulation in podocytes under LG or HG conditions by western blot. P values were determined by Student’s t -test and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (J) Assessment of nuclear ATF4 protein levels in cultured podocytes transfected with TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreated with 4-PBA under HG conditions for 48 h. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; * P < 0.01 ( n = 3 independent experiments).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot, Cell Culture, Transfection, shRNA, Real-time Polymerase Chain Reaction

Blockade of TLR4/MyD88/PKCδ/SHP-1 signaling attenuated podocyte cytoskeletal remodeling and inflammation, and macrophage recruitment and infiltration through decreasing ER stress and MCP-1 production under hyperglycemic conditions. (A) Assessment of podocyte migration by transwell assay in cultured podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-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 < 0.01; #### P < 0.01 ( n = 3 independent experiments). (B) Representative IF images by phalloidin staining to illustrate F-actin distribution and cytoskeletal remodeling in cultured podocytes treated with LG or HG 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). (C) Assessment of the secretion of inflammatory factors IL-6 and IL-1β in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (D) IHC staining using anti-f4/80 antibodies to illustrate macrophage infiltration in the glomeruli from mice at 16 weeks (Red arrow). Scale bar, 20 μm. ( n = 8) (E) The model diaphragm of podocyte-macrophage co-culture. (F) Representative images and assessment of macrophage migration by transwell assay after co-cultured with podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments).

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: Blockade of TLR4/MyD88/PKCδ/SHP-1 signaling attenuated podocyte cytoskeletal remodeling and inflammation, and macrophage recruitment and infiltration through decreasing ER stress and MCP-1 production under hyperglycemic conditions. (A) Assessment of podocyte migration by transwell assay in cultured podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-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 < 0.01; #### P < 0.01 ( n = 3 independent experiments). (B) Representative IF images by phalloidin staining to illustrate F-actin distribution and cytoskeletal remodeling in cultured podocytes treated with LG or HG 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). (C) Assessment of the secretion of inflammatory factors IL-6 and IL-1β in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (D) IHC staining using anti-f4/80 antibodies to illustrate macrophage infiltration in the glomeruli from mice at 16 weeks (Red arrow). Scale bar, 20 μm. ( n = 8) (E) The model diaphragm of podocyte-macrophage co-culture. (F) Representative images and assessment of macrophage migration by transwell assay after co-cultured with podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments).

Techniques: Migration, Transwell Assay, Cell Culture, Transfection, Staining, Immunohistochemistry, Co-Culture Assay

Schematic diaphragm of TLR4/MyD88/PKCδ/SHP-1 signaling in podocytes in exposure of hyperglycemia. Upon the stimuli of hyperglycemia, TLR4/MyD88 signaling was activated in glomerular podocyte; and PKCδ was then phosphorylated and bound to SHP-1 to provoke downstream ER stress, leading to nuclear translocation of ATF4 and enhanced transcriptional activity of MCP-1, which exacerbated damage to SD proteins and disruption of the cytoskeletal structure, increased cell motility, promoted inflammation in podocytes, and finally induced local macrophage migration and infiltration.

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: Schematic diaphragm of TLR4/MyD88/PKCδ/SHP-1 signaling in podocytes in exposure of hyperglycemia. Upon the stimuli of hyperglycemia, TLR4/MyD88 signaling was activated in glomerular podocyte; and PKCδ was then phosphorylated and bound to SHP-1 to provoke downstream ER stress, leading to nuclear translocation of ATF4 and enhanced transcriptional activity of MCP-1, which exacerbated damage to SD proteins and disruption of the cytoskeletal structure, increased cell motility, promoted inflammation in podocytes, and finally induced local macrophage migration and infiltration.

Techniques: Translocation Assay, Activity Assay, Disruption, Migration

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