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atf4 immunofluorescence staining (Cell Signaling Technology Inc)

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Cell Signaling Technology Inc atf4 immunofluorescence staining
Atf4 Immunofluorescence Staining, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+atf4/us12600720-355-16-26
Average 86 stars, based on 1 article reviews

atf4 immunofluorescence staining - by Bioz Stars, 2026-09

86/100 stars

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Article Title: Pyra-metho-carnil disrupts cancer cell proteostasis and induces apoptosis by binding to KDEL receptors.
Article Snippet: The following primary antibodies were used in this study: anti-KDELR (Stress Marq, SMC-129), anti-FLAG (M2) (Sigma-Aldrich, F1804), antiGM130 (BD Biosciences, 610822), anti-XBP1 (Abcam, ab220783), antiPERK (Cell Signaling Technologies, #5683), anti-phosphorylatedeIF2alpha (Ser51) (Cell Signaling Technologies, #9721), anti-eIF2alpha (Cell Signaling Technologies, #9722), anti-ATF4 (Cell Signaling Technologies, #11815), anti-CHOP (Cell Signaling Technologies, #9895), anti-caspase-3 (Cell Signaling Technologies, #9665), anti-cleaved caspase3 (Cell Signaling Technologies, #9664), anti-PARP (Cell Signaling Technologies, #9542), and anti-actin (Sigma-Aldrich, A2066).

Sonication:

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation
Article Snippet: .. The chromatin was fragmented by sonication on wet ice, and enriched with anti-ATF4 (11815) (Cell Signaling Technology, MA, USA), anti-ATF6 (70B1413.1) (Novus Biologicals, CO, USA), or anti-sXBP-1 (40435) (Cell Signaling Technology, MA, USA) antibodies, or non-specific IgG antibodies (ab171870) (Abcam, MA, USA). ..

Western Blot:

Article Title: Gcn2 deficiency triggers anemia and hypoxic intolerance in zebrafish.
Article Snippet: .. Standard Western blotting was performed with antibodies shown below: anti-HO-1 (ADI-SPA-895-D, Enzo), anti-Ferritin (Cell Signaling Technology (CST), 3998), anti-Gpx4 (Santa Cruz Biotechnology, sc-166120), anti-P-eIF2α (CST, 3597), anti-eIF2α (CST, 9722), and anti-Atf4 (CST, 11815). .. For HeLa cell experiments, the following antibodies were used: anti-GCN2 (CST, 3302), anti-HIF1α (CST, 36169), anti-SLC3A2 (CST, 13180), and antiSLC7A11 (CST, 12691). β-actin (CST, 4967) was used as the reference protein.

Incubation:

Article Title: UPR/ATF4/Noxa pathway overactivation through SERCA2 inhibition or ONC201 treatment combined with ABT-737 triggers apoptosis in chemoresistant ovarian cancer cells and patient-derived tumor organoids.
Article Snippet: Immunoblotting: equal amounts of proteins were loaded on 4-15% SDS-PAGE precast gel and transferred onto a PDVF membrane (Bio-Rad). .. Membranes were blocked 1 h with 5% nonfat dry milk in 0.05% Tween-TBS 1X then incubated with the following primary antibodies: anti-ATF4 (#11815), anti-BAK (#3814), anti-BAX (#2774), anti-BCL-xL (#2764), anti-BIM (#2819), anti-BiP (#3177), anti-CASPASE 3 (#9662), anti-CASPASE 8 (#9496), anti-CHOP (#2895), antiDR5 (#8074), anti-MCL-1 (#5453), anti-PARP (#9542), anti-PUMA (#12450) (Cell Signaling Technology); anti-Noxa (#114C307) (Calbiochem); anti-Actin (#MAB1501) (Merck Millipore); antiTubulin (#T6199-100UL) (DM-labo) and anti-SERCA2 (#NB300-581) (Novus-Bio). .. After incubation with the appropriate horseradish peroxidase-conjugate anti-mouse (#NA931V) (Amersham) or anti-rabbit (#7074) (Cell Signaling Technology), revelation was performed using AR TI CL E IN P RE SS ARTICLE IN PRESS Clarity Western EleCtroLuminescence detection reagent (Bio-Rad) and signal was recorded using ImageQuant800 (Cytiva).

Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
Article Snippet: Electrophoresis was followed by transfer to 0.45 μm PVDF membranes (Merck, IPVH00010). .. Membranes were blocked in 5 % milk in TBS-T for 1 h at RT before incubation with the following primary antibodies at 4 ◦C overnight: AntieIF2α (Cell Signaling, #9722S: RRID: AB_2230924, 1:1000 dilution), Anti-Phospho-eIF2a (Ser51) (Cell Signaling, #3597, RRID: AB_390740, 1:1000 dilution), anti-RNF25 (Abcam, ab89281, RRID: AB_2042820, 1:1000 dilution), Anti-Vinculin (Santa Cruz, Sc73614, RRID: AB_1131294, 1:1000 dilution), anti-ZNF598 (Sigma-Aldrich, HPA041760, RRID: AB_10792490, 1:2500 dilution), antiRNF14/ARA54 (Santa Cruz, sc-376701, RRID: AB_11150281, 1:1000 dilution), anti-GAPDH (Santa Cruz, sc-47724, RRID: AB_627678, 1:1000 dilution), Anti-Flag (Sigma-Aldrich, F3165, RRID: AB_259529, 1:1000 dilution), anti-GCN1 (Abcam, ab86139, RRID: AB_1925025, 1:1000 dilution), anti-GCN2 (Santa Cruz, sc-374609, RRID: AB_10986130, 1:1000 dilution), Anti-phosphoGCN2 (Thr899) (Cell Signaling, #94668S,1:1000 dilution), anti-ASCC3 (Thermo, #A304014A, RRID: AB_2620362, 1:1000 dilution), anti-EDF1 (Abcam, ab174651, RRID: AB_2893192, 1:1000 dilution), anti-tubulin (Sigma-Aldrich, T6074, RRID: AB_477582, 1:2000 dilution), anti-SLFN11 (Santa-Cruz, sc-515071, RRID: AB_3097730, 1:1000 dilution), anti-ZAK (Bethyl, #A301-993A, RRID: AB_1576612, 1:1000 dilution), anti-ATF4 (Cell Signaling, #11815, RRID:AB_2616025, 1:1000 dilution), anti-DNMT1 (Cell Signaling, #5032, RRID: AB_10548197, 1:1000 dilution), Anti-phospho-p38 MAPK (Thr180/Tyr82) (Cell Signaling # 9216; RRID: AB_331296), Anti-Phospho-SAPK/JNK (Thr183/Tyr185) (G9) (Cell Signaling #9255; RRID: AB_2307321, 1:1000 dilution), anti-DNMT2 (Santa Cruz, sc365001, RRID: AB_10708139, 1:1000 dilution), Afterwards, membranes were washed with TBS-T and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT: Goat-anti-mouse Immunoglobulins/ HRP (Dako, cat#P0447, RRID: AB_2617137, 1:3000-5000 dilution), Swine-anti-rabbit Immunoglobulins/HRP (Dako, cat#P0399, RRID: AB_2617141, 1:3000-5000 dilution). .. Finally, membranes were imaged using the ChemiDoc MP Imaging System (Bio-Rad).

Image Search Results

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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atf4 immunofluorescence staining (Cell Signaling Technology Inc)

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