mouse monoclonal antibodies against atf6  (Cell Signaling Technology Inc)

Journal: BioMed Research International

Article Title: Inhibition of eIF2 α Dephosphorylation Protects Hepatocytes from Apoptosis by Alleviating ER Stress in Acute Liver Injury

doi: 10.1155/2020/2626090

Figure Lengend Snippet: Inhibition of eIF2 α dephosphorylation mitigates ER stress in response to CCl 4 injury. (a) Levels of intrahepatic ATF6, XBP1s, and CHOP in control, salubrinal, ISRIB, PBA, and CCl 4 groups. (b) Salubrinal, ISRIB, or (c) the overexpressed DnaJC3 altered the expression of intrahepatic ATF6, XBP1s, and CHOP which was determined by Western blot. (d) Immunohistochemistry staining of CHOP expression in the liver (magnification ×100). Representative blots and immunohistochemistry from four independent experiments are shown. Histograms represent mean ± SD of four independent experiments ( n = 8-10). ∗∗ P < 0.01 versus the control group. † P < 0.05, †† P < 0.01 versus the CCl 4 or AAV8+CCl 4 group.

Article Snippet: The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline with Tween 20 (TBST) and probed with mouse monoclonal antibodies against ATF6 (MA1-25358, Thermo Fisher Scientific, USA), β -actin (sc-58673, sc: Santa Cruz Biotechnology), CHOP (ab11419, ab: abcam), and PERK (sc-377400) and rabbit monoclonal antibodies against ATF4 (11815, Cell Signaling Technology), cleaved caspase-3 (9664, Cell Signaling Technology), DnaJC3 (MA5-14820, Thermo Fisher Scientific, USA), phosphorylated eIF2 α (p-eIF2 α , 3398, Cell Signaling Technology), phosphorylated PERK (p-PERK, MA5-15033, Thermo Fisher Scientific, USA), and XBP1s (83418, mouse: 55 kD, human: 60 kD, Cell Signaling Technology).

Techniques: Inhibition, De-Phosphorylation Assay, Control, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: BioMed Research International

Article Title: Inhibition of eIF2 α Dephosphorylation Protects Hepatocytes from Apoptosis by Alleviating ER Stress in Acute Liver Injury

doi: 10.1155/2020/2626090

Figure Lengend Snippet: Inhibition of eIF2 α dephosphorylation reduces TG-induced ER stress and apoptosis in LO2 cells. (a) p-PERK, p-eIF2 α , ATF4, ATF6, XBP1s, CHOP, and cleaved caspase-3 protein expression at 12, 24, and 48 h post-DMSO (control) or post-TG (0.5 μ mol/L) incubation in LO2 cells. (b) Viability of LO2 cells determined by MTS. (c) Bar chart representing the apoptotic index in LO2 cells determined by flow cytometry. (d) p-eIF2 α , ATF4, ATF6, XBP1s, CHOP, and cleaved caspase-3 protein expression in control (DMSO pretreatment for 2 h and DMSO incubation for 24 h), salubrinal (salubrinal pretreatment for 2 h and DMSO incubation 24 h), TG (DMSO pretreatment for 2 h and TG incubation 24 h), and salubrinal+TG (salubrinal pretreatment for 2 h and TG incubation 24 h) groups. (e) Viability of LO2 cells determined by MTS. (f) Apoptotic index in LO2 cells determined by flow cytometry. Representative blots from four independent experiments are shown. Histograms represent mean ± SD of four independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 versus the control group. † P < 0.05 versus the TG group.

Article Snippet: The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline with Tween 20 (TBST) and probed with mouse monoclonal antibodies against ATF6 (MA1-25358, Thermo Fisher Scientific, USA), β -actin (sc-58673, sc: Santa Cruz Biotechnology), CHOP (ab11419, ab: abcam), and PERK (sc-377400) and rabbit monoclonal antibodies against ATF4 (11815, Cell Signaling Technology), cleaved caspase-3 (9664, Cell Signaling Technology), DnaJC3 (MA5-14820, Thermo Fisher Scientific, USA), phosphorylated eIF2 α (p-eIF2 α , 3398, Cell Signaling Technology), phosphorylated PERK (p-PERK, MA5-15033, Thermo Fisher Scientific, USA), and XBP1s (83418, mouse: 55 kD, human: 60 kD, Cell Signaling Technology).

Techniques: Inhibition, De-Phosphorylation Assay, Expressing, Control, Incubation, Flow Cytometry

Journal: BioMed Research International

Article Title: Inhibition of eIF2 α Dephosphorylation Protects Hepatocytes from Apoptosis by Alleviating ER Stress in Acute Liver Injury

doi: 10.1155/2020/2626090

Figure Lengend Snippet: PERK knockdown aggravates TG-induced ER stress and apoptosis in LO2 cells. LO2 cells were pretreated with PERK shRNA or control RNA for 48 h and then incubated with or without TG (0.5 μ mol/L) for another 24 h. (a) Detected protein levels of PERK, p-PERK, p-eIF2 α , ATF4, ATF6, XBP1s, CHOP, and cleaved caspase-3 in control shRNA (control shRNA pretreatment and DMSO incubation), PERK shRNA (PERK shRNA pretreatment and DMSO incubation), control shRNA+TG (control shRNA pretreatment and TG incubation), and PERK shRNA+TG (PERK shRNA pretreatment and TG incubation) groups by Western blot. (b) Bar chart showing the cell viability among the different experimental groups as determined by the MTS assay. (c) Cell apoptotic index determined by flow cytometry. Representative blots from four independent experiments are shown. Histograms showing the mean ± SD of four independent experiments. ∗ P < 0.05 versus the control shRNA group. † P < 0.05 versus the control shRNA+TG group.

Article Snippet: The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline with Tween 20 (TBST) and probed with mouse monoclonal antibodies against ATF6 (MA1-25358, Thermo Fisher Scientific, USA), β -actin (sc-58673, sc: Santa Cruz Biotechnology), CHOP (ab11419, ab: abcam), and PERK (sc-377400) and rabbit monoclonal antibodies against ATF4 (11815, Cell Signaling Technology), cleaved caspase-3 (9664, Cell Signaling Technology), DnaJC3 (MA5-14820, Thermo Fisher Scientific, USA), phosphorylated eIF2 α (p-eIF2 α , 3398, Cell Signaling Technology), phosphorylated PERK (p-PERK, MA5-15033, Thermo Fisher Scientific, USA), and XBP1s (83418, mouse: 55 kD, human: 60 kD, Cell Signaling Technology).

Techniques: Knockdown, shRNA, Control, Incubation, Western Blot, MTS Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Feedback loop between hepatocyte nuclear factor 1α and endoplasmic reticulum stress mitigates liver injury by downregulating hepatocyte apoptosis

doi: 10.1038/s41598-022-15846-8

Figure Lengend Snippet: Knockdown of RELA inhibits TG-induced HNF1α expression in LO2 cells. LO2 cells were pretreated with control shRNA, ATF6 shRNA, or RELA shRNA for 48 h, and then incubated with or without TG (1.0 μmol/L) for 36 h: ( A ) bar chart and representative western blotting showing the alteration in ATF6 and HNF1α expression after ATF6 shRNA transfection in LO2 cells; ( B ) bar chart and representative Western blotting showing the alteration in RelA and HNF1α expression after RELA shRNA transfection in LO2 cells; ( C ) bar chart representing the impact of RELA knockdown on LO2 cell viability; and ( D ) bar chart representing the relative protein expression and representative western blotting between the different experimental groups. ** p < 0.01, versus the control (control shRNA, or control shRNA + DMSO) or TG group (control shRNA + TG).

Article Snippet: Following blocking, membranes were probed with mouse monoclonal antibodies against ATF6 (sc-166659, 1:1000, Santa Cruz Biotechnology), eIF2α (sc-133132, 1:1000), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; sc-365062, 1:1000), HNF1α (sc-393668, 1:1000), p-RelA (sc-166748, 1:1000), or RelA (sc-514451, 1:1000), rabbit monoclonal antibodies against ATF4 (11,815, 1:1000, Cell Signaling Technology), cleaved caspase-3 (9664, 1:1000, Cell Signaling Technology), GRP78 (ab108615, 1:10,000, abcam), XBP1s (40,435, 1:1000, Cell Signaling Technology), or p-eIF2α (3398, 1:1000, Cell Signaling Technology), or rabbit polyclonal antibody against caspase-12 (2202, 1:1000, Cell Signaling Technology), protein bands were detected with enhanced chemiluminescent and images were processed using Quantity One software (Bio-Rad, Hercules, CA, USA).

Techniques: Knockdown, Expressing, Control, shRNA, Incubation, Western Blot, Transfection

Journal: Scientific Reports

Article Title: Feedback loop between hepatocyte nuclear factor 1α and endoplasmic reticulum stress mitigates liver injury by downregulating hepatocyte apoptosis

doi: 10.1038/s41598-022-15846-8

Figure Lengend Snippet: shRNA sequences used in LO2 cells.

Article Snippet: Following blocking, membranes were probed with mouse monoclonal antibodies against ATF6 (sc-166659, 1:1000, Santa Cruz Biotechnology), eIF2α (sc-133132, 1:1000), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; sc-365062, 1:1000), HNF1α (sc-393668, 1:1000), p-RelA (sc-166748, 1:1000), or RelA (sc-514451, 1:1000), rabbit monoclonal antibodies against ATF4 (11,815, 1:1000, Cell Signaling Technology), cleaved caspase-3 (9664, 1:1000, Cell Signaling Technology), GRP78 (ab108615, 1:10,000, abcam), XBP1s (40,435, 1:1000, Cell Signaling Technology), or p-eIF2α (3398, 1:1000, Cell Signaling Technology), or rabbit polyclonal antibody against caspase-12 (2202, 1:1000, Cell Signaling Technology), protein bands were detected with enhanced chemiluminescent and images were processed using Quantity One software (Bio-Rad, Hercules, CA, USA).

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: 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