Journal: International Journal of Molecular Sciences

Article Title: Strontium Attenuates Hippocampal Damage via Suppressing Neuroinflammation in High-Fat Diet-Induced NAFLD Mice

doi: 10.3390/ijms241210248

Figure Lengend Snippet: Sr restrained the HFD-induced apoptosis by altering expression levels of proteins related to the ERS pathway. ( A ) Western blot analysis of caspase-3, GRP78, IRE1α, p-IRE1α, XBP1, eIF2α, p-eIF2α, ATF4, ATF6, CHOP, and β-actin. ( B – K ) Relative protein expression of caspase-3 ( B ), GRP78 ( C ), IRE1α ( D ), p-IRE1α ( E ), XBP1 ( F ), eIF2α ( G ), p-eIF2α ( H ), ATF4 ( I ), ATF6 ( J ), and CHOP ( K ) in the hippocampi of each group of mice was examined through Western blotting ( n = 6 per group). Data were normalized with respect to the band of β-actin: the expression of target protein = the intensity of target protein band/the intensity of β-actin band. Results are shown as the ratio of the experimental group to the control group, and the values of the control group were taken as 1. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Subsequently, the following primary antibodies were used to incubate the membranes overnight at 4 °C: rabbit anti- NF-κB (#8242), rabbit anti- p38 (#9212), rabbit anti- ERK (#9102), rabbit anti-phospho- ERK ( p-ERK , #4370), rabbit anti-phospho- p38 ( p-p38 , #4511), and anti- caspase-3 (#9662) (purchased from Cell Signaling Technology (Danvers, MA, USA)); mouse anti- ATF6 (EM1701-94) (purchased from Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China); rabbit anti- XBP1 (A1731), rabbit anti-phospho- NF-κB ( p- NF-κB , AP0475), rabbit anti- GRP78 (A0241), and mouse anti- β-actin (purchased from Wuhan ABclonal Technology Co., Ltd., Wuhan, China); rabbit anti- eIF2α (ab115822), rabbit anti- TLR4 (ab13556), and rabbit anti-phospho- eIF2α ( p-eIF2α , ab32157) (purchased from Abcam (Cambridge, MA, USA)); and rabbit anti- CHOP (BM4962), anti-phospho- IRE1α ( p-IRE1α , BM4444), rabbit anti- ATF4 (BM5179), and rabbit anti- IRE1α (A00683-1) (purchased from Wuhan BOSTER Biological Technology Co., Ltd., Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: eLife

Article Title: Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma

doi: 10.7554/eLife.55865

Figure Lengend Snippet:

Article Snippet: Antibody , p-IRE1 (rabbit polyclonal) , AbNova , PAB12435 , 1:100.

Techniques: Transfection, Construct, Recombinant, Bicinchoninic Acid Protein Assay, Isolation, Detection Assay, Electroporation

Journal: International Journal of Molecular Sciences

Article Title: Hydrogen Sulfide Attenuates Hydrogen Peroxide-Induced Injury in Human Lung Epithelial A549 Cells

doi: 10.3390/ijms20163975

Figure Lengend Snippet: Hydrogen peroxide (H 2 O 2 ) suppressed endogenous hydrogen sulfide (H 2 S) production and H 2 S-producing enzymes. ( A ) H 2 S production tested using a methylene blue assay. ( B ) Quantitative real-time PCR (qRT-PCR) assay results for cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE), and 3-mercapto-pyruvate sulfurtransferase (MPST) mRNA expression levels. ( C ) Western blot analysis of CBS, CSE, and MPST protein expression levels. ( D ) Quantitative analysis of CBS, CSE, and MPST band intensities. The experiments were repeated at least three times. The results are presented as the mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. corresponding control group; # p < 0.05, ## p < 0.01 H 2 O 2 group vs. H 2 S + H 2 O 2 group).

Article Snippet: Primary antibodies were caspase-3, cleaved-caspase-3, Bcl-2, Bax, GRP78, CHOP, IRE1, elF2α, ATF4, ATF6, p-38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT and p-AKT (Wanleibio, Shenyang, China), β-actin, MPST, p-IRE1 (Bioss, Beijing, China), CBS, CSE (Omnimabs, Alhambra, CA, USA), and p-elF2α (Abbkine, Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hydrogen Sulfide Attenuates Hydrogen Peroxide-Induced Injury in Human Lung Epithelial A549 Cells

doi: 10.3390/ijms20163975

Figure Lengend Snippet: Quantitative real-time PCR (qRT-PCR) primers used in the study.

Article Snippet: Primary antibodies were caspase-3, cleaved-caspase-3, Bcl-2, Bax, GRP78, CHOP, IRE1, elF2α, ATF4, ATF6, p-38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT and p-AKT (Wanleibio, Shenyang, China), β-actin, MPST, p-IRE1 (Bioss, Beijing, China), CBS, CSE (Omnimabs, Alhambra, CA, USA), and p-elF2α (Abbkine, Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction

Journal: Molecular Biology of the Cell

Article Title: Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

doi: 10.1091/mbc.E10-08-0724

Figure Lengend Snippet: Gipie regulates the interaction between GRP78 and IRE1, and also ER stress-induced apoptosis. (A) Gipie suppressed ER stress-induced phosphorylation of JNK but not that of elF2α. Nontransfected (control) HUVECs or cells transfected with either Gipie or V5 empty vector were treated with TG at 1 μmol/l for 8 h, and total cell lysates were subjected to Western blot analysis using the indicated antibodies. (B) Regulation of GRP78 interaction with IRE1 by Gipie. Total cell lysates and GRP78 immunoprecipitates from nontransfected HUVECs or cells transfected with either Gipie or V5 empty vector were subjected to Western blot analysis using the indicated antibodies. Note that the amount of IRE1 detected in the GRP78 immunoprecipitates was increased by the transfection of Gipie (asterisk). In the right panels, the amounts of IRE1, ATF6, and PERK detected in the GRP78 immunoprecipitates shown in the left panels were quantified by densitometric scanning, and the values are presented as the fold increase in expression relative to the control (* p < 0.05; **not significant [N.S.], t test). (C) Effects of Gipie on ER stress-induced expression of CHOP. Total cell lysates from HUVECs that had been transfected with either Gipie or V5 empty vector were subsequently treated with TG at 1 μmol/l for 8 h and analyzed by Western blotting using anti-CHOP antibody. In the bottom panel, the amounts of CHOP were quantified by densitometric scanning, and the values are presented as the fold increase in expression relative to the control. An asterisk indicates a significant difference (*p < 0.05, t test). (D) Effects of Gipie on ER stress-induced apoptosis. HUVECs transfected with either Gipie or V5 empty vector were incubated with TG at 1 μmol/l for 8 h, followed by annexin V staining (green) to detect apoptotic cells. In the right panel, the percentage of annexin V–positive cells was quantified. An asterisk indicates a statistically significant difference (* p < 0.05, t test). (E and F) Effects of endogenous Gipie depletion on JNK phosphorylation (E) and the interaction between GRP78 and IRE1 (F) Nontransfected HUVECs or cells transfected with either control or Gipie-specific siRNA were collected. Total cell lysates and GRP78 immunoprecipitates were subjected to Western blot analysis using the indicated antibodies. In the bottom panel of (F), the amounts of IRE1 detected in the GRP78 immunoprecipitates were quantified by densitometric scanning, and the values are presented as the fold increase in expression relative to the control. An asterisk indicates statistically significant difference (* p < 0.05, t test). (G) Effects of Gipie-depletion on ER stress-induced expression of CHOP. Total cell lysates from HUVECs that had been transfected with either control or Gipie-specific siRNA were analyzed. An asterisk indicates a statistically significant difference (* p < 0.05, t test).

Article Snippet: Other antibodies used in this study include anti-GRP78 and anti-KDEL mouse monoclonal antibodies (mAbs) (Stressgene, Victoria, BC, Canada); anti-GRP78, anti-PERK, and anti-PDI rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDI and anti-CHOP mouse mAbs (Affinity BioReagents, Golden, CO); anti-GM130 and PECAM-1 mouse mAbs (BD Bioscience PharMingen, San Diego, CA); anti-IRE1 mouse mAb (MoBiTec GmbH, Gottingen, Germany); anti-ATF6 rabbit polyclonal antibody (LifeSpan Bioscience, Seattle, WA); anti-PERK and anti-PDI rabbit polyclonal antibodies (Santa Cruz Biotechnology); anti-eIF2α mouse mAb, anti–phospho-eIF2α (Ser51) rabbit mAb, anti-JNK, anti–phospho-JNK (Thr183/Tyr185) rabbit polyclonal antibodies, and anti-CHOP mouse mAb (Cell Signaling Technology, Beverly, MA); anti–α-SMA and anti–β-actin mouse mAbs (Sigma-Aldrich, St. Louis, MO); and anti-HaloTag rabbit polyclonal antibody (Promega).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Incubation, Staining

Journal: Molecular Biology of the Cell

Article Title: Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

doi: 10.1091/mbc.E10-08-0724

Figure Lengend Snippet: Proposed model of Gipie-mediated modulation of the UPR pathway in endothelial cells. In cells exposed to persistent ER stresses, the expression of Gipie was induced to regulate the interaction of GRP78 and IRE1, resulting in a protective role against apoptotic cell death by regulating JNK activation and ER stress-induced expression of CHOP.

Article Snippet: Other antibodies used in this study include anti-GRP78 and anti-KDEL mouse monoclonal antibodies (mAbs) (Stressgene, Victoria, BC, Canada); anti-GRP78, anti-PERK, and anti-PDI rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); anti-PDI and anti-CHOP mouse mAbs (Affinity BioReagents, Golden, CO); anti-GM130 and PECAM-1 mouse mAbs (BD Bioscience PharMingen, San Diego, CA); anti-IRE1 mouse mAb (MoBiTec GmbH, Gottingen, Germany); anti-ATF6 rabbit polyclonal antibody (LifeSpan Bioscience, Seattle, WA); anti-PERK and anti-PDI rabbit polyclonal antibodies (Santa Cruz Biotechnology); anti-eIF2α mouse mAb, anti–phospho-eIF2α (Ser51) rabbit mAb, anti-JNK, anti–phospho-JNK (Thr183/Tyr185) rabbit polyclonal antibodies, and anti-CHOP mouse mAb (Cell Signaling Technology, Beverly, MA); anti–α-SMA and anti–β-actin mouse mAbs (Sigma-Aldrich, St. Louis, MO); and anti-HaloTag rabbit polyclonal antibody (Promega).

Techniques: Expressing, Activation Assay