Journal: World Journal of Gastroenterology

Article Title: Mesalazine alleviated the symptoms of spontaneous colitis in interleukin-10 knockout mice by regulating the STAT3/NF-κB signaling pathway

doi: 10.3748/wjg.v31.i7.96459

Figure Lengend Snippet: Effect of mesalazine on alleviating endoplasmic reticulum stress in interleukin-10 -/- mouse colonic epithelial cells. A: Transmission electron microscope photograph of mouse colonic epithelial cells (magnification × 8200); B: Glucose-regulated protein 78 protein level; C: Representative images of glucose-regulated protein 78 immunofluorescence (green) and nuclei (blue) in colonic tissue; D: Representative images of C/EBP homologous protein immunofluorescence (green) and nuclei (blue) in colonic tissue. Compared with the interleukin-10 -/- group, a P < 0.05, b P < 0.01. WT: Wild type; IL: Interleukin; ASA: Amino salicylic acid; GRP78: Glucose-regulated protein 78; DAPI: 4’,6-diamidino-2-phenylindole; CHOP: C/EBP homologous protein.

Article Snippet: They were incubated overnight at 4 °C with rabbit anti-GRP78 polyclonal antibodies (1:100, 11587-1-AP, Proteintech) and rabbit anti-CHOP antibodies (1:100, 5554S, Cell Signaling Technology).

Techniques: Transmission Assay, Microscopy, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pyrotinib targeted EGFR/GRP78 mediated cell apoptosis in high EGFR gene copy number gastric cancer

doi: 10.1186/s13046-025-03485-6

Figure Lengend Snippet: Pyrotinib drives EGFR translocated to ER and interacted with GRP78. A . Lysates from SNU719 and NUGC4 cells treated with DMSO or Pyr (1 µM) were precipitated with EGFR antibodies by immunoprecipitation. The precipitated proteins were assayed by western blot. B - C . Representative images ( B ) and quantification ( C ) of the proximity ligation assay (PLA) detecting EGFR-GRP78 interactions in SNU719 and NUGC4 cells treated with DMSO or Pyr (1µM). Red dots indicate PLA signals. Scale bar, 40 μm. Significance between groups was determined by One-way ANOVA. * p < 0.05; **p < 0.01; *** p < 0.001. D . Immunofluorescence analysis of ER-tracker (red), GRP78 (green), and EGFR (cyan) in SNU719 cells treated with Pyr (1µM). Scale bar, 50 μm. E . HEK293T cells were transfected with the indicated plasmid, treated with or without Pyr (1µM), followed by immunoprecipitation with anti-flag antibody, and eventually assayed by WB analysis with the indicated antibodies. F - G . SNU719 ( F ) and NUGC4 ( G ) cells were transfected with flag-GRP78 and treated with or without Pyr (1 µM) for 48 h. Cell lysates were subjected to immunoprecipitation using an anti-Flag antibody, followed by western blot analysis with the indicated antibodies. H . HEK293T cells were co-transfected with GRP78-flag and EGFR-HA (FL: full length, or its truncation mutants) plasmids for 48 h, followed by co-immunoprecipitation (CO-IP) assay. I . HEK293T cells were co-transfected with EGFR-HA and GRP78-FLAG (FL or its truncation mutants) plasmids for 48 h and treated with Pyr (1 µM) for 48 h, followed by CO-IP assay

Article Snippet: Antibodies against rabbit CHOP (15204-1-AP), mouse GRP78 (for immunofluorecence, 66574-1-Ig) were purchased from Proteintech.

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Immunofluorescence, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pyrotinib targeted EGFR/GRP78 mediated cell apoptosis in high EGFR gene copy number gastric cancer

doi: 10.1186/s13046-025-03485-6

Figure Lengend Snippet: Pyrotinib accelerated the ubiquitination of GRP78 via blocking Thr62 phosphorylation. A . CO-IP assays were performed to assess GRP78 phosphorylation in SNU719 cells treated with EGF stimulation (100 ng/mL) for 5 min, followed with or without treatment of Pyr for 24 h. The lysates were incubated with indicated antibodies. B . CO-IP assays were performed to detect the phosphorylation of GRP78 in EGFR-overexpressing or/and GRP78-overexpressing SNU719 cells and HEK-293T cells after stimulating with EGF (100 ng/mL) for 5 min. C . Mutational analysis of GRP78 residues (T62A, T69A, T534A) identified critical phosphorylation sites mediating Pyr-induced GRP78 dephosphorylation. D . Western blot analysis revealed dose-dependent reductions in GRP78 protein levels after 24 h treatment with increasing Pyr concentrations in SNU719 cells. E . Cycloheximide (CHX, 100 µg/mL) chase experiments demonstrated accelerated GRP78 degradation in Pyr (1 µM, 24 h) treated versus untreated SNU719 cells. F . SNU719 cells co-transfected with Ub-HA and GRP78-Flag plasmids were treated with Pyr (1 µM) and MG132 (10 µM). Immunoblotting confirmed enhanced GRP78 ubiquitination under proteasomal inhibition. G . HEK293T cells were co-transfected with the indicated plasmids for 48 h and then treated with MG132 (10 µM). The polyubiquitination levels of GRP78 protein were analyzed. H . HEK293T cells expressing GRP78-Flag with WT ubiquitin or mutant (Ub-K48O, Ub-K63O) plasmids were treated with Pyr (1 µM, 24 h). Ubiquitination patterns confirmed K48-linked polyubiquitination as the dominant degradation signal. I . Immunoprecipitation was performed in SNU719 lysates transfected with flag-GRP78 using Flag antibodies, and the precipitants were measured by western blotting with the indicated antibodies. J . Immunoprecipitation was performed in SNU719 lysates transfected with HA-TRIM21 using HA antibodies, and the precipitants were measured by western blotting with the indicated antibodies. K . SNU719 cells co-transfected with indicated plasmids and/or si TRIM21 , then treated with or without Pyr (1 µM). WB analysis showed reduced polyubiquitination of the si TRIM21 group compared to control. L . HEK293T cells co-transfected with indicated plasmids and treated with MG132 (10 µM) showed reduced polyubiquitination of the T62D mutant compared to WT GRP78 upon Pyr (1 µM, 24 h) stimulation. Significance between groups was determined by ( E , I ) Paired t-test. * p < 0.05; **p < 0.01; *** p < 0.001

Article Snippet: Antibodies against rabbit CHOP (15204-1-AP), mouse GRP78 (for immunofluorecence, 66574-1-Ig) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Blocking Assay, Phospho-proteomics, Co-Immunoprecipitation Assay, Incubation, De-Phosphorylation Assay, Western Blot, Transfection, Inhibition, Expressing, Mutagenesis, Immunoprecipitation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pyrotinib targeted EGFR/GRP78 mediated cell apoptosis in high EGFR gene copy number gastric cancer

doi: 10.1186/s13046-025-03485-6

Figure Lengend Snippet: Pyrotinib enhances oxaliplatin chemosensitivity through GRP78 inhibition. A . Western blot analysis revealed differential expression of DNA double-strand break repair proteins (γ-H2A.X) and GRP78 in NUGC4 and SNU719 cells treated with DMSO, Pyr (1 µM), or/and OXA (2.5 µM) for 24 h. B . The expression of GRP78 protein in SNU719 and NUGC4 cells transfected with si NC or si GRP78 . C . CCK-8 cytotoxicity assays demonstrated enhanced OXA sensitivity in GRP78 silenced SNU719 and NUGC4 cells. D . The expression of GRP78 protein in SNU719 and NUGC4 cells transfected with MOCK or OE GRP78 plasmid. E . GRP78 overexpressing GC cells exhibited OXA resistance in CCK-8 assays, showing higher viability than control cells. F - G . Immunofluorescence staining ( F ) and analysis ( g ) showed that after stimulation by OXA (2.5 µM) for 24 h, the γ-H2A.X foci in SNU719 and NUGC4 cell nucleus distinguished in MOCK and OE GRP78 groups. Blue background indicates DAPI, red dots indicate γ-H2A.X foci. Bars, 40 μm. H . Schematic diagram display that Pyr promotes EGFR-GRP78 complex formation in the ER lumen, subsequently activating the PERK/eIF2α/ATF4/CHOP signaling pathway, and finally resulted in the tumor apoptosis in EGFR-high CN GC. Besides, Pyr could sensitize GC cells to OXA by inhibiting GRP78 phosphorylation at T62 which results in GRP78 ubiquitination. Significance between groups was determined by ( C , E ) Paired t-test and ( G ) One-way ANOVA. * p < 0.05; **p < 0.01; *** p < 0.001

Article Snippet: Antibodies against rabbit CHOP (15204-1-AP), mouse GRP78 (for immunofluorecence, 66574-1-Ig) were purchased from Proteintech.

Techniques: Inhibition, Western Blot, Quantitative Proteomics, Expressing, Transfection, CCK-8 Assay, Plasmid Preparation, Control, Immunofluorescence, Staining, Phospho-proteomics, Ubiquitin Proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: LC-MS/MS results obtained from the protein band corresponding to the XAP-1 antigen. (A) The MS/MS spectrum of a peptide from the LC-MS/MS analysis. It contains product ions of the b- and y- series that matched peptide sequence ITPSYVAFTPEGER from Grp78 from Mus musculus. (B) The m/z ratios for theoretical product ions of the y- and b- series. Product ions that were observed in the MS/MS spectrum are shown in bold.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Protein sequence of Mus musculus Grp78. The peptide from our MS/MS analysis is shown in gray text and represents 2.14% coverage.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Depletion of IgM and Grp78 from the outer segment–enriched preparation. IgM and Grp78 were depleted from outer segment (OS) cell extracts. IgM and the XAP-1 antigen are shown in red, whereas Grp78 is shown in green. Overlap of the signals (E, F, yellow). A small amount of IgM was detected after one round of depletion, but none remained after a second round (A). Both the XAP-1 antigen (B) and Grp78 (C, D) remained in the sample after all IgM was removed. The XAP-1 antigen and Grp78 overlap at an identical relative molecular mass (F). Depletion of Grp78 (C, D) abolished the signal obtained with the XAP-1 antibody (B).

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Immunohistochemical localization of Grp78 in the retinas of mouse and frog. After antigen retrieval, retinas from C57BL6/J mice were immunostained using anti-Grp78 (A, green), PNA (B, red), and iodide (C, blue). (C) Composite image. Yellow (arrows) indicates areas of overlap of the Grp78 and PNA, indicating that in the outer retina, Grp78 is localized to cone photoreceptors in a pattern identical with that of the XAP-1 antigen. (C, inset) Higher magnification image of the area within the inset in C. In the mouse, Grp78 is also found in all cell layers of the retina in a location corresponding to ER. (D) Examination of the frog retina revealed a different pattern. The peripheries of both rod and cone photoreceptor outer segments and the inner segments are immunolabeled in this species. Scale bar, 10 μm. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Immunohistochemical staining, Immunolabeling

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation

Journal: Scientific reports

Article Title: PERK/EIF2AK3 integrates endoplasmic reticulum stress-induced apoptosis, oxidative stress and autophagy responses in immortalised retinal pigment epithelial cells.

doi: 10.1038/s41598-022-16909-6

Figure Lengend Snippet: Figure 1. PERK downregulation increases ER stress. PERK signalling pathway mediates induction of ER stress in ARPE-19 cells treated with 1 μg/ml BFA through increased expression of GRP78 (a), PERK (b), p-eif2α (Ser51)/eif2α (c) and CHOP (d); representative immunoblots shown at 24-h and 48-h time-points (e). BFA- induced ER stress in cells in which PERK is downregulated with siRNA leads to significant upregulation of GRP78 (f) at 24-h time point and significant downregulation of PERK pathway components PERK (g), p-eif2α (Ser51)/eif2α (h) and CHOP (i) at 24 and 48-h time points when compared to non-targeting control siRNA; (i) representative immunoblots shown. Phosphorylation of PERK was observed from the slower migration of a total PERK band at 170 kDa following treatment with BFA (b,g,e,j). Phosphorylation of eif2α at serine 51 (p-eif2α (Ser 51)) was calculated relative to total eif2a protein expression (c,h). Results are presented as fold- changes of the 0-h time point (a–d) or the NT group in cells transfected with non-targeting control siRNA (f–i). Protein levels were normalised to α-tubulin. Means ± standard deviation are shown (n = 3). Statistical analysis was performed using a one-way ANOVA with Dunnet’s post-hoc test (a–d) or two-way ANOVA with Sidak’s multiple-comparison test (f–i) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). BFA Brefeldin A, NT non- treated, NS non-significant.

Article Snippet: Antibody Supplier (code) Description PERK Abcam (ab65142) Rabbit polyclonal eif2α Cell signalling technology (#9722) Rabbit polyclonal eif2α pSer51 Cell signalling technology (#9721) Rabbit polyclonal GRP78 (BiP) Cell signalling technology (#3177) Rabbit monoclonal Nrf2 pSer40 Abcam (ab76026) Rabbit monoclonal NQO1 Cell signalling technology (#3187) Mouse monoclonal SQSTM1/p62 Cell signalling technology (#5114) Rabbit polyclonal LC3B Cell signalling technology (#2775) Rabbit polyclonal CHOP Cell signalling technology (#2895) Mouse monoclonal Bax (D2E11) Cell signalling technology (#5023) Rabbit monoclonal Bcl2 Abcam (ab32124) Rabbit monoclonal Alpha Tubulin Abcam (ab4074) Rabbit polyclonal Table 2.

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Migration, Transfection, Standard Deviation, Comparison