mab against atf6  (Novus Biologicals)

Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: Huh7.5 cells were treated with DMSO or 10 µM IXA4 for 72 hours. (A) Cellular RNA was harvested and XBP1s (IRE1 pathway), CHOP (PERK pathway) and HERPUD1 (ATF6 pathway) mRNA expression was assessed by RT qPCR. (B) Expression of genes related to LD biogenesis ( FASN , DGAT1 and DGAT2 ) was assessed by RT-qPCR. (C) Huh7 cells were electroporated with 5 µg of HCV SGR RNA. Four hours-post electroporation, cells were treated with DMSO or 10 µM IXA4 for 72h and RNA replication was measured by luciferase expression. Data are expressed as a percentage of HCV replication in DMSO-treated samples. (D-E) Huh7.5 cells were pre-treated with DMSO or 0.05 µM Tg for 30 minutes, and subsequently infected with HCV at MOI 0.2 for 72h. (D) Cells were fixed and stained for cell nuclei (DAPI, blue) and LDs (BODIPY-493; green), HCV core (red), and assessed by confocal microscopy under 63x magnification. (E) Cellular supernatant was collected, and viral extracellular titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Expressing, Quantitative RT-PCR, Electroporation, Luciferase, Infection, Staining, Confocal Microscopy, Focus Forming Assay

Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: (A-C) Huh7.5 cells were transduced with shCTRL, shATF6, shIRE1 or shPERK lentivirus to generate stable knockdowns of ATF6, IRE1 and PERK. (A) Successful silencing was confirmed by western blot analysis. (B-C) Cells were treated with DMSO or 0.1 µM Tg for 30 minutes and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (B) Cells were fixed and stained for cell nuclei (DAPI; blue), lipid droplets (BODIPY-493; green) or HCV core (red) and imaged using a confocal microscope at 63x magnification. (C) Cellular supernatants were collected, and viral titer was assessed by focus-forming assay. (D) Huh7.5 cells were treated with DMSO or 1 µg/mL tunicamycin (Tm) for 4 hours. Cellular RNA was harvested and XBP1s and HERPUD1 mRNA expression was assessed by RT-qPCR. (E-G) Huh7.5 cells were pre-treated with DMSO or 1 µg/mL Tm for four hours, and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (E) intracellular HCV RNA was assessed by RT-qPCR, (F) Cells were fixed and stained as described in (B) . (G) Viral titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Transduction, Western Blot, Infection, Staining, Microscopy, Focus Forming Assay, Expressing, Quantitative RT-PCR, Confocal Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APC Cdc20 .

doi: 10.1002/advs.202405441

Figure Lengend Snippet: Figure 1. TPD52 promotes ATF6 activation during the UPR. a) Anti-TPD52 immunoprecipitates (IPs) coupled with mass spectrometry analysis (MS) to identify TPD52-interacting proteins in T24 cells. IPs-MS results were subjected to enrichment analysis. b) The proteins were enriched in the “protein processing in endoplasmic reticulum” categories. c) Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-TPD52 immunoprecipitates (IPs)

Article Snippet: Rabbit primary antibodies against ATF6 (65 880), eIF2α (5324), phospho-eIF2α (Ser 51) (3398), PERK (3192), IRE1α (3294), Bip (3177), cyclin D1 (2978), Flag-tag (14 793), HA-tag (3724), GSTtag (2625) and β-actin (4970) were all purchased from Cell Signaling Technology, Inc (Boston, MA, USA).

Techniques: Activation Assay, Mass Spectrometry, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APC Cdc20 .

doi: 10.1002/advs.202405441

Figure Lengend Snippet: Figure 2. TPD52 regulates UPR signals and ER size through ATF6. a) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from T24, 5637, and A375 cells stably expressing shTPD52 or Scr. Scr, Scramble. b) IB analysis of WCL derived from 253J, J82, and SW780 cells stably overexpressing TPD52 or EV. EV, empty vector. c) IB analysis of the WCL derived from wild-type (WT) or Tpd52-knockout (TPD52−/−) mice bladder tissues. d) Representative transmission electron micrographs (TEMs) image of T24 cells stably expressing shTPD52 or Scr. Where indicated, 1 μg mL−1 Tu was added before harvesting the cells. Scr, Scramble. Scale bars: 500 nm (white); 200 nm (red). e) ER thickness was quantified for the indicated TEM samples in (d). f) Spearman analysis of gene-expression data from patients with bladder cancer (BLCA, TCGA dataset, n = 411 samples) and skin cutaneous melanoma (SKCM, TCGA dataset, n = 472 samples) was used for depicting the correlation between TPD52 and unfolded protein response (UPR). g) IB analysis of WCL derived from 253J ATF6 knockdown or control cells transduced with TPD52 lentivirus or T24 TPD52 knockdown or control cells transduced with ATF6 lentivirus. TPD, TPD52. Scr, Scramble. EV, empty vector. h) Representative TEMs image of T24 ATF6 knockdown or control cells transduced with TPD52 lentivirus. Where indicated, 1 μg mL−1 Tu was added before harvesting the cells. Scr, Scramble. EV, empty vector. Scale bars: 500 nm (white); 200 nm (red). i. ER thickness was quantified for the indicated TEM samples in (h).

Article Snippet: Rabbit primary antibodies against ATF6 (65 880), eIF2α (5324), phospho-eIF2α (Ser 51) (3398), PERK (3192), IRE1α (3294), Bip (3177), cyclin D1 (2978), Flag-tag (14 793), HA-tag (3724), GSTtag (2625) and β-actin (4970) were all purchased from Cell Signaling Technology, Inc (Boston, MA, USA).

Techniques: Western Blot, Derivative Assay, Stable Transfection, Expressing, Plasmid Preparation, Knock-Out, Transmission Assay, Gene Expression, Knockdown, Control, Transduction

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: List of shRNA sequences.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: shRNA, Sequencing, Control

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: ATF6 silencing aggravates TG-induced necroptosis and ER stress and reduces RIP3 expression in LO2 cells. LO2 cells were infected with control shRNA or ATF6 shRNA for 48 h, and then they were incubated with DMSO or TG (0.5 μ mol/L) for another 24 h: (a) comparison of cell viability between the control group (control shRNA + DMSO), the ATF6 shRNA group (ATF6 shRNA + DMSO), the TG group (control shRNA + TG), and the ATF6 shRNA + TG group in LO2 cells; (b) bar chart representing the ATF6, RIP3, and p-MLKL protein expression and representative western blotting analyzing the protein expression among the different experimental groups; (c) bar chart representing CHOP protein expression and representative western blotting evaluating the protein expression among the TG group and the ATF6 shRNA + TG group. ∗ p < 0.05 and ∗∗ p < 0.01 versus the control group or the TG group.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: Expressing, Infection, Control, shRNA, Incubation, Comparison, Western Blot

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: RIP3 silencing alleviates TG-induced necroptosis and ER stress in LO2 cells. LO2 cells were infected with control shRNA or RIP3 shRNA for 48 h, and then they were incubated with DMSO or TG (0.5 μ mol/L) for another 24 h: (a) comparison of cell viability between the control group (control shRNA + DMSO), the RIP3 shRNA group (RIP3 shRNA + DMSO), the TG group (control shRNA + TG), and the RIP3 shRNA + TG group in LO2 cells; (b) bar chart representing the RIP3 and p-MLKL protein expression and representative western blotting analyzing the protein expression among the different experimental groups; (c) bar chart representing CHOP protein expression and representative western blotting demonstrating the protein expression among the TG group and the ATF6 shRNA + TG group. ∗∗ p < 0.01 versus the control group or the TG group.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: Infection, Control, shRNA, Incubation, Comparison, Expressing, Western Blot

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: Induction of acute liver injury in mice. Male BALB/c mice were administered olive oil (CCl 4 solvent), PBS (TM solvent), CCl 4 , or TM for 12, 24, 48 h ( n = 12): (a) enzymatic rate method to detect the time-dependent changes of serum ALT levels in the CCl 4 -induced liver injury mouse model; (b) serum TBil levels measured using the diazonium method in the different experimental groups; (c) H&E staining representing pathological changes in liver tissue and bar charts representing the proportion of necrotic liver tissue area; (d) protein expression of intrahepatic RIP3, CHOP, ATF6, and p-MLKL measured by western blotting after CCl 4 injection. ∗∗ p < 0.01 versus the olive oil group and ## p < 0.01 versus the PBS group.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: Solvent, Staining, Expressing, Western Blot, Injection

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: Atf6 knockdown aggravates liver injury and ER stress and reduces RIP3 expression in CCl 4 -induced mice. Mice were pretreated with control shRNA or Atf6 shRNA for 6 weeks, then they were injected with olive oil or CCl 4 for 24 h ( n = 12): (a) the enzymatic rate method demonstrating the changes of serum ALT levels in the control group (control shRNA + olive oil), the Atf6 shRNA group (Atf6 shRNA + olive oil), the CCl 4 group (control shRNA + CCl 4 ), and the Atf6 shRNA + CCl 4 group; (b) serum TBil levels measured using the diazonium method in the different experimental groups; (c) H&E staining representing pathological changes in liver tissue and bar charts representing the proportion of necrotic liver tissue area; (d) western blot analysis of intrahepatic ATF6, RIP3, and p-MLKL expression among the different experimental groups; (e) qPCR analysis demonstrating the relative ATF6 and RIP3 expression among the different experimental groups; (f) western blot analysis of intrahepatic caspase-12 and CHOP expression level in the CCl 4 group and the Atf6 shRNA + CCl 4 group. ∗∗ p < 0.01 versus the control shRNA group or the control shRNA + CCl 4 group.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: Knockdown, Expressing, Control, shRNA, Injection, Staining, Western Blot

Journal: BioMed Research International

Article Title: Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

doi: 10.1155/2021/8717565

Figure Lengend Snippet: RIP3 shRNA reduces hepatocyte necroptosis. ATF6 plays multiple roles in acute liver injury and plays a dominant role in protecting the liver. It reduces hepatocyte necroptosis through a negative feedback regulation of ER stress. It also upregulates RIP3, which is not favorable to the recovery process. On the other hand, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress.

Article Snippet: Following blocking, membranes were incubated with mouse monoclonal antibodies against ATF6 (sc-166659, 1 : 1000, Cell Signaling Technology, USA), CHOP (GADD153, sc-71136, 1 : 10000, Santa Cruz Biotechnology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062, 1 : 1000, Santa Cruz Biotechnology), RIP3 (sc-374639, 1 : 1000, Santa Cruz Biotechnology, USA), rabbit monoclonal antibodies, phosphorylated MLKL (p-MLKL, 37333S, 1 : 1000, Cell Signaling Technology, USA), MLKL (PA5-34733, 1 : 1000, Thermo Fisher Scientific, USA), or rabbit polyclonal antibodies against caspase-12 (2202, 1 : 1000, Cell Signaling Technology).

Techniques: shRNA

Journal: Scientific Reports

Article Title: Mitochondrial Dysregulation Secondary to Endoplasmic Reticulum Stress in Autosomal Dominant Tubulointerstitial Kidney Disease – UMOD (ADTKD- UMOD )

doi: 10.1038/srep42970

Figure Lengend Snippet: ( A ) ADTKD- UMOD is characterized by maturation and trafficking defect of mutant UMOD and intracellular accumulation of UMOD in TAL cells. UMOD immunolocalization revealed a diffuse cytoplasmic staining with enforcement of the luminal membrane in TAL cells of a wild-type mouse. In contrast, TAL cells of an Umod C93F mutant mouse displayed a strong paranuclear immunopositivity for UMOD. Wild-type: Umod wt mouse; Umod C93F : homozygous Umod C93F mutant mouse. Age of mice analysed: four months. Chromogen: DAB, nuclear staining: haemalum. ( B ) Heat map of relative expression values (z scores) showed differential abundance of several proteins localized in the ER. ( C ) In the outer medulla of Umod mutant mice of both mouse lines, a strong accumulation of immature UMOD was present. ( D ) Protein abundances of BiP, phospho-eIF2α, eIF2α, ATF4, both full-length (§) and cleaved activated (#) ATF6, and CHOP were increased in Umod mutant mice compared to wild-type mice. Signal intensities were corrected for GAPDH signal intensities of the same PVDF-membrane, which was stripped several times to facilitate the detection of multiple proteins. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type) = 1]. Data are shown as means ± SD. One-way ANOVA with Newman-Keuls’s post hoc test: p vs. wild-type, *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: rabbit monoclonal antibody against phospho-AMPKα (Thr172) (40H9, no. 2535, Cell Signaling), rabbit polyclonal antibody against AMPKα (no. 2532, Cell Signaling), rabbit monoclonal antibody against ATF4 (D4B8, no. 11815, Cell Signaling), mouse monoclonal antibody against ATF6 (70B1413.1, no. NBP1-40256, novusbio), rabbit monoclonal antibody against ATM (D2E2, no. 2873, Cell Signaling), rabbit polyclonal antibody against BiP (no. 3183, Cell Signaling), rabbit monoclonal antibody against CHOP (D46F1, no. 5554, Cell Signaling), rabbit polyclonal antibody against FIS1 (no. GTX111010, GeneTex), rabbit monoclonal antibody against GAPDH (D16H11, no. 5174, Cell Signaling), rabbit monoclonal antibody against LC3A (D50G8, no. 4599, Cell Signaling), rabbit monoclonal antibody against LC3B (D11, no. 3868, Cell Signaling), rabbit monoclonal antibody against phospho-LKB1 (Ser428) (C67A3, no. 3482, Cell Signaling), rabbit monoclonal antibody against LKB1 (D60C5, no. 3047, Cell Signaling), rabbit polyclonal antibody against NRF1 (no. 12381, Cell Signaling), rabbit polyclonal antibody against PGC-1α (no. NBP1-04676, novusbio), mouse monoclonal antibody against PGC-1β (no. sc-373771, Santa Cruz), rabbit monoclonal antibody against SDHA (D6J9M, no. 11998, Cell Signaling), and rabbit polyclonal antibody against human THP (H-135, no. sc-20631, Santa Cruz Biotechnology).

Techniques: Mutagenesis, Staining, Membrane, Expressing, Quantitative Proteomics

Journal: Journal of Endocrinology

Article Title: Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors

doi: 10.1530/joe-12-0311

Figure Lengend Snippet: Figure 2 Exendin-4 inhibits HG/PA-induced expression of ER stress markers. (A) INS-1 cells were treated with vehicle (CON) or HG/PA in the absence or presence of 5 nM exendin-4 (Ex) for 6 h. Total RNA was isolated and mRNA expression of sXBP1, CHOP, and Bip were determined by qRT-PCR. Cyclophilin was used as control gene. (B) INS-1 cells were treated as described earlier for 12 or 18 h and total cell extracts were analyzed for PERK, p-PERK, p-eIF2a, p-IRE1a, p-JNK, ATF6 (p50), and actin by immunoblotting using specific antibodies. (C) Quantitative analysis of western blots. Relative abundance of each band was estimated by densitometric analysis. Each bar represents the meanGS.E.M. from three independent experiments. *P!0.05.

Article Snippet: The sources of various reagents and materials were as follows: exendin-4, D-glucose, palmitate (PA), fatty acid-free BSA were from Sigma–Aldrich; anti-C/EBPb and anti-lamin B antibodies were from Santa Cruz Biotechnology, Inc.; polyclonal antibody against SREBP1 (SREBF1) was from Abcam (Cambridge, MA, USA); MAB against ATF6 was from IMGENEX(SanDiego,CA,USA); andanti-C/EBPhomologous protein (CHOP), anti-caspase-3, anti-poly(ADP-ribose) polymerase (PARP), anti-PERK, anti-phospho-PERK, anti-phosphoIRE1a, anti-phospho-c-Jun N-terminal kinase (JNK), and antiphospho-eukaryotic initiation factor 2a (eIF2a) antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Western Blot