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Proteintech rabbit anti gapdh
Rabbit Anti Gapdh, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+atf4/ATF4+Antibody/pm41931883-64-28-31
Average 96 stars, based on 651 article reviews

rabbit anti gapdh - by Bioz Stars, 2026-11

96/100 stars

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Article Title: LPS–TLR4 signaling attenuates CHOP-mediated apoptosis under endoplasmic reticulum stress conditions during porcine embryonic development
Article Snippet: A total of 80–100 embryos per group were lysed in RIPA buffer with protease/phosphatase inhibitors (Thermo Fisher Scientific) and boiled at 95 °C for 10 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. .. After blocking with 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies such as rabbit anti-ATF4 (1:1000, 10835-1-AP, Proteintech), rabbit anti-CHOP (1:1000, SC-166682; Santa Cruz Biotechnology), rabbit anti-phospho-eIF2α (1:1000, AP0341; ABclonal), rabbit anti- LC3 (1:800, ab58610; Abcam), rabbit anti-TLR4 (1:1000, 19811-1-AP, Proteintech), and rabbit anti-GAPDH (1:1000, sc-365062, Santa Cruz Biotechnology). ..

Article Title: Pan-viral ORFs discovery using Massively Parallel Ribosome Profiling
Article Snippet: .. All membranes were then incubated for 16 h at 4 ̊C in one of the following primary antibodies diluted in blocking buffer: rabbit anti-ATF4 (Proteintech 10835-1-AP, 1:1000), mouse anti-EIF2ɑ (Abcam ab5369, 1:1000), rabbit anti-phospho(Ser51)-EIF2ɑ (Abcam ab32157, 1:2000), rabbit anti-PKR (Abcam ab32052, 1:1000), rabbit anti-phospho(Thr451)-PKR (Abcam ab81303, 1:1000), rabbit antiSTAT1 (Proteintech 10144-2-AP, 1:2000), mouse anti-phospho(Tyr701)-STAT1 (BD Biosciences 612233, 1:1000), rabbit anti-IRF3 (Cell Signaling 11904, 1:1000), rabbit antiphospho(Ser386)-IRF3 (Abcam ab76493, 1:2000), and vinculin (R&D Systems MAB6896, 2 μg/mL). .. Total protein concentrations were determined using the Pierce 660 protein assay reagent (Thermo Fisher, 22660) or BCA protein assay kit (Thermo Fisher, 23225).

Incubation:

Article Title: Organelle stress in pyometra: Molecular evidence of endoplasmic reticulum stress and mitochondrial dysfunction.
Article Snippet: This study aimed to explore the key molecular changes related to endoplasmic reticulum stress and mitochondrial damage in the uterine tissues of bitches with open pyometra.. A total of 16 bitches were included in the study, divided into a healthy control group (n = 8) and a pyometra group (n = 8).. The results showed that compared with healthy individuals, the mRNA expression. and protein abundance of endoplasmic reticulum stress marker genes PERK, ATF4, CHOP, GRP78 and GRP94 were significantly upregulated in the uterine tissues of bitches with pyometra.

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(A) Glioma cell survival at 24 h after SS treatment. (B) Glioma cell survival after pretreatment with DFO, FAC, Fer‐1, and Lip‐1, respectively, for 1 h before SS treatment. (C–F) Intracellular ROS, MDA, GSH/GSSG, and ferrous ion levels at 6, 12, and 24 h after SS treatment, with and without DFO, Fer‐1, Lip‐1, and FAC pretreatment for 1 h before SS treatment, respectively. (G) Glioma cell survival after SS treatment, with and without pretreatment with NAC and GSH for 1 h before SS treatment, respectively. (H) Western blots of ferroptosis‐related protein expression after SS treatment. Cell survival was assessed by the CCK‐8 assay. (I) Western blots of ATF3 expression in the cytoplasm and nuclei of SS‐treated cells. (J) Viability of cells treated with siATF3 followed by SS. (K–N) Ferrous ion, MDA, GSH/GSSG, and ROS levels in cells treated with siATF3 followed by SS. (O) Confocal microscopic images of ATF3 immunofluorescence in U251 cells treated with SS. (P, Q) Confocal microscopy images and fluorescence intensity measurements of mitochondrial superoxide in SS‐ and siATF3‐treated U251 and T98 cells. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Journal: CNS Neuroscience & Therapeutics

Article Title: ATF3 Promotes Solasonine‐Triggered Ferroptosis in Glioma Cells by Suppressing Glycolysis

doi: 10.1002/cns.71226

Figure Lengend Snippet: (A) Glioma cell survival at 24 h after SS treatment. (B) Glioma cell survival after pretreatment with DFO, FAC, Fer‐1, and Lip‐1, respectively, for 1 h before SS treatment. (C–F) Intracellular ROS, MDA, GSH/GSSG, and ferrous ion levels at 6, 12, and 24 h after SS treatment, with and without DFO, Fer‐1, Lip‐1, and FAC pretreatment for 1 h before SS treatment, respectively. (G) Glioma cell survival after SS treatment, with and without pretreatment with NAC and GSH for 1 h before SS treatment, respectively. (H) Western blots of ferroptosis‐related protein expression after SS treatment. Cell survival was assessed by the CCK‐8 assay. (I) Western blots of ATF3 expression in the cytoplasm and nuclei of SS‐treated cells. (J) Viability of cells treated with siATF3 followed by SS. (K–N) Ferrous ion, MDA, GSH/GSSG, and ROS levels in cells treated with siATF3 followed by SS. (O) Confocal microscopic images of ATF3 immunofluorescence in U251 cells treated with SS. (P, Q) Confocal microscopy images and fluorescence intensity measurements of mitochondrial superoxide in SS‐ and siATF3‐treated U251 and T98 cells. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Article Snippet: Transferrin receptor (TfR), transferrin, ferroportin (FPN), ferritin, GPX4, xCT, H2A, ß‐actin, ATG5, NCOA4, ferritin heavy chain (FTH), microtubule‐associated protein 1 light chain 3ß (LC3B), p62, pyruvate kinase M2 (PKM2), phosphofructokinase platelet type (PFKP), catalase, glucose‐regulated protein 78 (GRP78), protein kinase RNA–like endoplasmic reticulum kinase (PERK), ATF4, ATF3, and hexokinase 2 (HK2) antibodies were purchased from Abclonal (China).

Techniques: Western Blot, Expressing, CCK-8 Assay, Confocal, Immunofluorescence, Microscopy, Fluorescence, Control

(A) Western blots of ferritinophagy‐related protein expression in glioma cells after SS treatment. (B–E) Cell viability and ROS, MDA, and ferrous ion levels after pretreatment with 3MA and Baf‐A1 followed by SS treatment. (F) Western blots of ferroptosis‐ and ferritinophagy‐related protein expression in glioma cells after 3MA and Baf‐A1 pretreatment and SS treatment. (G) Viability of glioma cells treated with siATG5 followed by SS. (H–J) ROS, MDA, and ferrous ion levels in glioma cells treated with siATG5 followed by SS. (K, L) Western blots of ferritinophagy‐ and ferroptosis‐related protein expression in glioma cells treated with siATG5 and siATF3, respectively, followed by SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Journal: CNS Neuroscience & Therapeutics

Article Title: ATF3 Promotes Solasonine‐Triggered Ferroptosis in Glioma Cells by Suppressing Glycolysis

doi: 10.1002/cns.71226

Figure Lengend Snippet: (A) Western blots of ferritinophagy‐related protein expression in glioma cells after SS treatment. (B–E) Cell viability and ROS, MDA, and ferrous ion levels after pretreatment with 3MA and Baf‐A1 followed by SS treatment. (F) Western blots of ferroptosis‐ and ferritinophagy‐related protein expression in glioma cells after 3MA and Baf‐A1 pretreatment and SS treatment. (G) Viability of glioma cells treated with siATG5 followed by SS. (H–J) ROS, MDA, and ferrous ion levels in glioma cells treated with siATG5 followed by SS. (K, L) Western blots of ferritinophagy‐ and ferroptosis‐related protein expression in glioma cells treated with siATG5 and siATF3, respectively, followed by SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Techniques: Western Blot, Expressing, Control

(A) Western blots of antioxidant‐related protein expression after SS treatment. (B, C) The intracellular hydrogen peroxide level and antioxidant‐related protein expression after pretreatment with SP and ATP, respectively, followed by SS treatment. (D) Ferrous ion levels after hydrogen peroxide treatment. (E, F) Western blots of ferritinophagy‐related protein expression in hydrogen peroxide– and NAC‐treated cells. (G–I) ATP, PA, and hydrogen peroxide levels in glioma cells treated with siATF3 and SS. (J) Western blots of glycolysis‐related protein expression in glioma cells treated with siATF3 and SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Journal: CNS Neuroscience & Therapeutics

Article Title: ATF3 Promotes Solasonine‐Triggered Ferroptosis in Glioma Cells by Suppressing Glycolysis

doi: 10.1002/cns.71226

Figure Lengend Snippet: (A) Western blots of antioxidant‐related protein expression after SS treatment. (B, C) The intracellular hydrogen peroxide level and antioxidant‐related protein expression after pretreatment with SP and ATP, respectively, followed by SS treatment. (D) Ferrous ion levels after hydrogen peroxide treatment. (E, F) Western blots of ferritinophagy‐related protein expression in hydrogen peroxide– and NAC‐treated cells. (G–I) ATP, PA, and hydrogen peroxide levels in glioma cells treated with siATF3 and SS. (J) Western blots of glycolysis‐related protein expression in glioma cells treated with siATF3 and SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Techniques: Western Blot, Expressing, Control

(A–C) Western blots of ER stress–related protein expression after SS treatment with and without SP and ATP pretreatment, and after hydrogen peroxide treatment. (D–F) ROS, MDA, and ferrous ion levels in cells treated with SS after pretreatment with 4‐PBA. (G, H) Western blots of ferroptosis‐ and ER stress–related protein expression in cells treated with 4‐PBA and SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Journal: CNS Neuroscience & Therapeutics

Article Title: ATF3 Promotes Solasonine‐Triggered Ferroptosis in Glioma Cells by Suppressing Glycolysis

doi: 10.1002/cns.71226

Figure Lengend Snippet: (A–C) Western blots of ER stress–related protein expression after SS treatment with and without SP and ATP pretreatment, and after hydrogen peroxide treatment. (D–F) ROS, MDA, and ferrous ion levels in cells treated with SS after pretreatment with 4‐PBA. (G, H) Western blots of ferroptosis‐ and ER stress–related protein expression in cells treated with 4‐PBA and SS. * p < 0.01 versus control, # p < 0.01 versus treatment group.

Techniques: Western Blot, Expressing, Control

(A) Photograph of nude mouse tumors. (B) Tumor volume over time. (C–F) Hydrogen peroxide, MDA, ferrous ion, and GSH/GSSG levels in tumor tissues from nude mice treated with high and low doses of SS. (G) Western blots of glycolysis‐, ferritinophagy‐, and ferroptosis‐related protein expression in tumor tissues. (H) H&E staining of intracranial orthotopic tumor following SS treatment. (I) Expression of intratumoral NCOA4, PKM2, and GPX4 in each treatment group (bar = 50 μm). * p < 0.01 versus control.

Journal: CNS Neuroscience & Therapeutics

Article Title: ATF3 Promotes Solasonine‐Triggered Ferroptosis in Glioma Cells by Suppressing Glycolysis

doi: 10.1002/cns.71226

Figure Lengend Snippet: (A) Photograph of nude mouse tumors. (B) Tumor volume over time. (C–F) Hydrogen peroxide, MDA, ferrous ion, and GSH/GSSG levels in tumor tissues from nude mice treated with high and low doses of SS. (G) Western blots of glycolysis‐, ferritinophagy‐, and ferroptosis‐related protein expression in tumor tissues. (H) H&E staining of intracranial orthotopic tumor following SS treatment. (I) Expression of intratumoral NCOA4, PKM2, and GPX4 in each treatment group (bar = 50 μm). * p < 0.01 versus control.

Techniques: Western Blot, Expressing, Staining, Control

Schematic illustration of the fabrication and application of the WT-Exo–loaded coaxial nanofibrous membrane for rescuing p75NTR deficiency–associated osteogenic impairment and promoting bone regeneration.

Journal: Materials Today Bio

Article Title: EMSCs-derived exosomes rescue p75NTR −/− -mediated osteogenesis deficiency via PI3K/Akt/ERK1/2 signaling pathways

doi: 10.1016/j.mtbio.2026.103755

Figure Lengend Snippet: Schematic illustration of the fabrication and application of the WT-Exo–loaded coaxial nanofibrous membrane for rescuing p75NTR deficiency–associated osteogenic impairment and promoting bone regeneration.

Article Snippet: Osteogenesis-related antibodies, including anti-Col-I (A5786), anti-OPN (A1499), anti-activating transcription factor 4 (ATF4, A18687) and anti-ALP (A0514), were obtained from Abclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Membrane

Gene expression profiles between (SF/PCL 2:5 /PVA) WT-Exo and SF/PCL 2:5 /PVA groups. (A) Volcano plot displaying differentially expressed genes between the (SF/PCL 2:5 /PVA) WT-Exo and SF/PCL 2:5 /PVA groups. Red dots represent upregulated genes, and blue dots represent downregulated genes. (B) Heatmap of selected differentially expressed genes. (C) KEGG pathway enrichment analysis. (D) Gene Ontology (GO) analysis showing enriched biological processes, cellular components and molecular functions. (E) Venn diagram showing the overlap of differentially expressed genes annotated to the PI3K/Akt signaling pathway, ERK1/2 signaling cascade, and osteoblast differentiation–related gene sets. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Journal: Materials Today Bio

Article Title: EMSCs-derived exosomes rescue p75NTR −/− -mediated osteogenesis deficiency via PI3K/Akt/ERK1/2 signaling pathways

doi: 10.1016/j.mtbio.2026.103755

Figure Lengend Snippet: Gene expression profiles between (SF/PCL 2:5 /PVA) WT-Exo and SF/PCL 2:5 /PVA groups. (A) Volcano plot displaying differentially expressed genes between the (SF/PCL 2:5 /PVA) WT-Exo and SF/PCL 2:5 /PVA groups. Red dots represent upregulated genes, and blue dots represent downregulated genes. (B) Heatmap of selected differentially expressed genes. (C) KEGG pathway enrichment analysis. (D) Gene Ontology (GO) analysis showing enriched biological processes, cellular components and molecular functions. (E) Venn diagram showing the overlap of differentially expressed genes annotated to the PI3K/Akt signaling pathway, ERK1/2 signaling cascade, and osteoblast differentiation–related gene sets. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Techniques: Gene Expression, Analysis, Significance Assay

Journal: Materials Today Bio

Article Title: EMSCs-derived exosomes rescue p75NTR −/− -mediated osteogenesis deficiency via PI3K/Akt/ERK1/2 signaling pathways

doi: 10.1016/j.mtbio.2026.103755

Figure Lengend Snippet:

Techniques:

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).

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

Further validation of PKCδ activation being indispensable for ER stress and MCP-1 production through SHP-1 in diabetic podocyte injury. (A) Schematic representation of PKCδ plasmids to express PKCδ proteins in DN (tyrosine mutation at amino acid 311), CA or WT form. (B) Representative western blot to validate the expression of DN-PKCδ, CA-PKCδ and WT-PKCδ plasmids in HEK-293 T cells by using antibodies specific for HA-tag, with GAPDH detection as a loading control. ( n = 3 independent experiments). (CDE) Determination of protein levels of SHP-1 (C), ER stress proteins BIP, sXBP-1, ATF4 and ATF6 (D), and MCP-1 (E) in cultured mouse podocytes transfected with DN-, CA- and WT-PKCδ plasmids, or control plasmids under LG or HG conditions. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; # P < 0.05; ## 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: Further validation of PKCδ activation being indispensable for ER stress and MCP-1 production through SHP-1 in diabetic podocyte injury. (A) Schematic representation of PKCδ plasmids to express PKCδ proteins in DN (tyrosine mutation at amino acid 311), CA or WT form. (B) Representative western blot to validate the expression of DN-PKCδ, CA-PKCδ and WT-PKCδ plasmids in HEK-293 T cells by using antibodies specific for HA-tag, with GAPDH detection as a loading control. ( n = 3 independent experiments). (CDE) Determination of protein levels of SHP-1 (C), ER stress proteins BIP, sXBP-1, ATF4 and ATF6 (D), and MCP-1 (E) in cultured mouse podocytes transfected with DN-, CA- and WT-PKCδ plasmids, or control plasmids under LG or HG conditions. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Techniques: Biomarker Discovery, Activation Assay, Mutagenesis, Western Blot, Expressing, Control, 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

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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