Journal: Oncogene

Article Title: GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer.

doi: 10.1038/onc.2012.212

Figure Lengend Snippet: Figure 1. CLU associates with GRP78 under ER stress condition. (a) Immunoblotting following IP confirmed the presence of GRP78, 94 and 170 in CLU co-IP. LNCaPCLU-2 cells were treated with 200 nM thapsigargin (TG) for 12 h. Pre-cleared whole cell lysates were extracted for co-IP with goat anti-CLU polyclonal antibody and detected with the indicated antibodies. Both vinculin and ponseau staining for IgG heavy chain served as loading controls. (b) LNCaPCLU-2 cells were treated with 200 nM TG for 12 h and then co-immunostained with anti-GRP78 (red) or CLU (green) antibodies. Images were acquired using Zeiss confocal microscope. Objectives, 40 oil. Scale bar, 10 mm. (c) LNCaPCLU-2 cells were further transfected with mock or GRP78 and then treated with 10 mM MG132 for 24 h. Pre-cleared whole cell lysates were extracted for co-IP with goat anti-CLU or GRP78 polyclonal antibody and detected with mouse anti-CLU monoclonal or goat anti-GRP78 polyclonal antibody. Representative result is from at least three independent experiments. Co-IP, co-immunoprecipitation; DMSO, a solvent control; psCLU, presecretary CLU; sCLU, secreted CLU; TG, tharpsigargin; WCL, whole cell lysates.

Article Snippet: Human GRP78 (HSPA5; NM_005347.2) cDNA (Origene, Rockville, MD, USA) was used for further subcloning into pHR–CMV lenti-viral vector; 50- or 30- flanking BamHI or XhoI restriction enzyme sites were generated using PCR with appropriate primers: forward, 50-GGATCCATGAAGCTCTCCCTGGTG-30 , reverse, 50-CTCGAGATCAGTGTCTACAACTCATC-30 .

Techniques: Western Blot, Co-Immunoprecipitation Assay, Staining, Microscopy, Transfection, Immunoprecipitation, Solvent, Control

Journal: Oncogene

Article Title: GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer.

doi: 10.1038/onc.2012.212

Figure Lengend Snippet: Figure 2. ER stress increases cytoplasmic CLU and GRP78 protein levels, while suppressing secretion of sCLU. (a) Upper panel, LNCaPCLU-2 cells were treated with 200 nM thapsigargin (TG) or 10 mM MG132 for indicated hours. DMSO was kept the same as the maximum time with the stressors, 12 h for TG and 24 h for MG132. 20 mg of whole cell lysates were resolved by SDS–PAGE and detected with appropriated antibodies. Hypoglycosylated psCLU and sCLU proteins are indicated by bars. Lower panel, culture media was replaced 24 h after cells were plated, and sCLU in the culture media was measured using ELISA. sCLU concentration (ng/ml) was normalized by live cell numbers. Compared to untreated cells, **Po0.01. (b) Upper panel, six N-glycoyslation sites were mutated at aspargines (N) 86, 103, 145, 291, 354 and 374 to glutamine (Q) that are equally distributed in CLU b and a chain. Lower panel, LNCaP cells were transfected with CLUWT, CLUNbQ or CLUNaQ. 20 mg of whole cell lysates and 20 ml culture media were subjected to SDS–PAGE followed by western blot with CLU antibody. Hypoglycosylated psCLU and sCLU proteins are indicated by bars. Vinculin or ponceau staining was used as loading controls. Representative results from at least three independent experiments. CLUNaQ, CLU a chain with another three substitutions of N for Q at 291, 354 and 374; CLUNbQ, CLU b chain with three substitutions of N for Q at 86, 103 and 145; CLUWT, wild-type CLU; Cul Med, culture media; WCL, whole cell lysates.

Article Snippet: Human GRP78 (HSPA5; NM_005347.2) cDNA (Origene, Rockville, MD, USA) was used for further subcloning into pHR–CMV lenti-viral vector; 50- or 30- flanking BamHI or XhoI restriction enzyme sites were generated using PCR with appropriate primers: forward, 50-GGATCCATGAAGCTCTCCCTGGTG-30 , reverse, 50-CTCGAGATCAGTGTCTACAACTCATC-30 .

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Western Blot, Staining

Journal: Oncogene

Article Title: GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer.

doi: 10.1038/onc.2012.212

Figure Lengend Snippet: Figure 4. GRP78 enhances CLU retrotranslocation and redistribution to the mitochondria. (a) LNCaP cells were transfected with mock, CLU, GRP78 or co-transfected with both CLU and GRP78 plasmids for 48 h, followed by 10 nM paclitaxel treatment for additional 24 h. A 50-mg of cytosolic, microsomal or mitochondrial proteins were resolved by SDS–PAGE and western blot analysis performed with appropriate antibodies. The same blot was stripped and immunoblotted with vinculin (cytosol), calregulin (microsome) and Tim23 (mitochondria) antibodies to show the purity of fractions. Hypoglycosylated psCLU and sCLU proteins are indicated by bars. Cyto, cytosolic; Micro, microsomal; Mito, mitochondrial. (b) Real-time monitoring CLUGFP (green) and mitochondria (red) dynamics. Live cells were labeled with a mitochondria probe, MitoTracker Deep Red FM (ex.644/ em.665) and visualized in red (Mito). Arrowheads indicate migration of CLUGFP to the mitochondria. (c) Cells were pre-labeled with Mitotracker and treated with 100 nM paclitaxel for 6 h. CLU–mitochondria colocalization was determined by the Mander’s coefficient after 1801 rotation along the y axis of a 3D-reconstruction of a z axis stack (nX3, 40 cells per experiment). Imaging and 3D reconstructions with volume rendering of the stacks were performed with the Zeiss 780M confocal microscope and Zen 2010 software. Objectives, 60 oil. Scale bar, 5 mm. *Po0.05, **Po0.01.

Article Snippet: Human GRP78 (HSPA5; NM_005347.2) cDNA (Origene, Rockville, MD, USA) was used for further subcloning into pHR–CMV lenti-viral vector; 50- or 30- flanking BamHI or XhoI restriction enzyme sites were generated using PCR with appropriate primers: forward, 50-GGATCCATGAAGCTCTCCCTGGTG-30 , reverse, 50-CTCGAGATCAGTGTCTACAACTCATC-30 .

Techniques: Transfection, SDS Page, Western Blot, Labeling, Migration, Imaging, Microscopy, Software

Journal: Oncogene

Article Title: GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer.

doi: 10.1038/onc.2012.212

Figure Lengend Snippet: Figure 6. GRP78 knockdown enhances paclitaxel chemosensitivity. Left panel, PC3 cells were transfected with scramble siRNA or GRP78 siRNA for 48 h, followed the treatment with 20 nM paclitaxel for additional 48 h. A 50-mg of whole cell lysates were resolved by SDS–PAGE and immunoblotted with indicated antibodies. Right panel, live cell numbers were counted by trypan blue staining. Compared to Scr, ***Po0.001. Lipo, lipofectamine; Scr, scramble siRNA; siGRP78, siRNA against GRP78; Tax, paclitaxel; UT, untreated.

Article Snippet: Human GRP78 (HSPA5; NM_005347.2) cDNA (Origene, Rockville, MD, USA) was used for further subcloning into pHR–CMV lenti-viral vector; 50- or 30- flanking BamHI or XhoI restriction enzyme sites were generated using PCR with appropriate primers: forward, 50-GGATCCATGAAGCTCTCCCTGGTG-30 , reverse, 50-CTCGAGATCAGTGTCTACAACTCATC-30 .

Techniques: Knockdown, Transfection, SDS Page, Staining

Journal: Brain : a journal of neurology

Article Title: GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

doi: 10.1093/brain/awz168

Figure Lengend Snippet: Figure 4 The identification of GRP78 as the target antigen of LEMS-PCD IgG using immunoprecipitation. (A) Proteins isolated from TY10 cells were separated by the gel, transferred onto the membranes and analysed by western blotting. A strong protein band of 80 kDa in molecular weight reacted with one PCD-LEMS patient (Patient 3) and one LEMS patient (Patient 9). (B) Living cell-based antibody binding assay. Patient or control IgG bound and crosslinked to the cell surface of living TY10 cells. After the cells were lysed and the protein A/G beads were incubated with them, antibody-bound proteins were separated using protein A/G beads, eluted with DTT and assessed for GRP78 by western blot. Bands for the variant form of GRP78, corresponding to 60 kDa, were detected in antibody-bound protein lysates from PCD-LEMS IgG (Patients 2–5), LEMS IgG (Patient 9), but it was not observed from the other LEMS IgG (Patients 1 and 6–8) or eight control IgGs. Pt. = Patient.

Article Snippet: For immunoprecipitation, 125 or 62.5 mg/ml PCD-LEMS IgG (Patients 3 and 5) or LEMS IgG (Patient 9) was incubated with 5 mg HEK 293 T cell lysates with or without the overexpression of FLAG-tagged GRP78 (Origene) for 4 h at 4 C, and then incubated with 40 ml anti-FLAG-IgG coupling resin (EZview Red Anti-FLAG M2 Affinity Gel beads; SigmaAldrich) for 2 h at 4 C. After the immune complexes were pulled down, the supernatants were saved for analysis.

Techniques: Immunoprecipitation, Isolation, Western Blot, Molecular Weight, Binding Assay, Control, Incubation, Variant Assay

Journal: Brain : a journal of neurology

Article Title: GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

doi: 10.1093/brain/awz168

Figure Lengend Snippet: Figure 5 Detection of GRP78 antibodies in LEMS-PCD patients by the living cell-based antibody binding assay. A single band for splicing the variant form of GRP78, corresponding to 60 kDa, was detected in antibody-bound protein lysates from 11/14 (78%) PCD-LEMS IgGs, not in those from LEMS IgGs (0%: 0/10), in the living cell-based antibody binding assay by western blot. GRP78 recombinant protein and TY cell lysate was used as positive control (P.C).

Article Snippet: For immunoprecipitation, 125 or 62.5 mg/ml PCD-LEMS IgG (Patients 3 and 5) or LEMS IgG (Patient 9) was incubated with 5 mg HEK 293 T cell lysates with or without the overexpression of FLAG-tagged GRP78 (Origene) for 4 h at 4 C, and then incubated with 40 ml anti-FLAG-IgG coupling resin (EZview Red Anti-FLAG M2 Affinity Gel beads; SigmaAldrich) for 2 h at 4 C. After the immune complexes were pulled down, the supernatants were saved for analysis.

Techniques: Binding Assay, Variant Assay, Western Blot, Recombinant, Positive Control

Journal: Brain : a journal of neurology

Article Title: GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

doi: 10.1093/brain/awz168

Figure Lengend Snippet: Figure 6 The effect of removal of GRP78-specific IgG from PCD-LEMS IgG on NF-B p65 nuclear translocation in TY10 cells. (A) The number of NF-B p65 nuclear-positive TY10 cells was compared after exposure to PCD-LEMS IgG [Patient 3 (125 mg/ml, 62.5 mg/ml), Patient 5 (125 mg/ml), Patient 9 (125 mg/ml)] immunoadsorbed against GRP78 or control protein. The data represent the means from two independent experiments that were performed in triplicate. Statistical significance was determined by a paired two-tailed t-test. (B) The binding of LEMS-PCD IgG to BMECs after immunoadsorption of GRP78-specific IgG were decreased compared to that without removal of GRP78 antibodies. Cells were incubated with LEMS-PCD IgG, and then labelled with Alexa Fluor 488 goat anti-human IgG (green). Images were captured by an IN Cell Analyzer 2000 (GE healthcare) at 20 magnification. Scale bar = 200 mm. Pt. = Patient.

Article Snippet: For immunoprecipitation, 125 or 62.5 mg/ml PCD-LEMS IgG (Patients 3 and 5) or LEMS IgG (Patient 9) was incubated with 5 mg HEK 293 T cell lysates with or without the overexpression of FLAG-tagged GRP78 (Origene) for 4 h at 4 C, and then incubated with 40 ml anti-FLAG-IgG coupling resin (EZview Red Anti-FLAG M2 Affinity Gel beads; SigmaAldrich) for 2 h at 4 C. After the immune complexes were pulled down, the supernatants were saved for analysis.

Techniques: Translocation Assay, Control, Two Tailed Test, Binding Assay, Incubation

Journal: The American journal of pathology

Article Title: Epithelial calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.

doi: 10.1016/j.ajpath.2013.07.014

Figure Lengend Snippet: Figure 3 Calreticulin (CRT) overexpression in tubular epithelial cells (TECs) induces endoplasmic reticulum (ER) stress and cellular apoptosis mechanisms. A: Representative blot shows increased GRP78 expression, an endoplasmic reticulum stress sensor, in CRT-overexpressing cells. Quanti- fication corresponds to means SD of three independent experiments. TUNEL-positive nucleieDAPI ratio (B) and cleaved caspase-3eDAPI ratio (C) in control and CRT-overexpressing cells under normal growth conditions or after treatment with 50 mmol/L H2O2 for 2 hours. The number of DAPI- positive cells counted for each cell line is indicated above the respective graph bars. Data represent means SD of three independent experiments. D: Representative blot shows increased GRP78 expression in CRT knockdown cells. Quantification corresponds to means SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control.

Article Snippet: The primary antibodies used were CRT (catalog No. 06- 661; Upstate Biotechnology, Inc., Lake Placid, NY), Ecadherin (BD 610181; BD Biosciences, San Jose, CA), vinculin (Sigma V9131; Sigma), vimentin (catalog No. MS-129; Thermo Scientific, Inc., Waltham, MA), GRP78 (AP06149PU-N; Acris Antibodies GmbH, Herford, Germany), fibronectin (sc-8422; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), COL1A1 (sc-25974; Santa Cruz Biotechnology), phospho-Smad3 (ab52903; Abcam, Cambridge, England), Smad3 (catalog No. 3102; Cell Signaling Technology, Inc., Danvers, MA), b-actin (catalog No. A5316; Sigma), and b-tubulin (ab6046; Abcam).

Techniques: Over Expression, Expressing, TUNEL Assay, Control, Knockdown

Journal: The American journal of pathology

Article Title: Epithelial calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.

doi: 10.1016/j.ajpath.2013.07.014

Figure Lengend Snippet: Figure 6 Increased cellular stress characterizes calreticulin (CRT)- overexpressing tubular cells in vivo. Representative double immunostaining images of GRP78 (green) and CRT (red) in control (sham-operated) mice and at 2 or 8 days after ureteric ligation in study animals (L2 and L8, respectively). Both GRP78 and CRT are expressed at low levels in control animals but are gradually increased and highly co-localized in the tubular cells of the fibrotic kidney after 2 and 8 days of UUO (yellow), indicating that CRT-overexpressing tubular cells in vivo exhibit broad ER stress. Scale bars: 50 mm.

Article Snippet: The primary antibodies used were CRT (catalog No. 06- 661; Upstate Biotechnology, Inc., Lake Placid, NY), Ecadherin (BD 610181; BD Biosciences, San Jose, CA), vinculin (Sigma V9131; Sigma), vimentin (catalog No. MS-129; Thermo Scientific, Inc., Waltham, MA), GRP78 (AP06149PU-N; Acris Antibodies GmbH, Herford, Germany), fibronectin (sc-8422; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), COL1A1 (sc-25974; Santa Cruz Biotechnology), phospho-Smad3 (ab52903; Abcam, Cambridge, England), Smad3 (catalog No. 3102; Cell Signaling Technology, Inc., Danvers, MA), b-actin (catalog No. A5316; Sigma), and b-tubulin (ab6046; Abcam).

Techniques: In Vivo, Double Immunostaining, Control, Ligation

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs induce ERS in HD11 cells to evade immune clearance by macrophages . Transmission electron microscopy shows ER network expansion and swelling in HD11 cells treated with WT, WTΔ ypjA , or OMVs. The data represent one of three independent experiments. Scale bar, 2 μm. B OMVs colocalized with ER. DiO-OMVs were incubated with HD11 cells at 37 °C for 6 h, the ER was labeled with ER-Tracker Red, and laser confocal microscopy imaging was performed. The data represent one of three independent experiments. Scale bar, 7.5 μm. Colocalization scatter plot was generated using ImageJ software and GraphPad Prism 8. C HD11 cells were treated with 50–200 µg/mL OMVs for 6 h, and the relative mRNA level of GRP78/BiP was determined by qPCR. ( n = 3) D HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and the relative mRNA level of GRP78/BiP was determined by qPCR. ( n = 3) E , F HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and western blot was used to detect the expression of GRP78/BiP protein in cell lysates and gray value analysis was performed using ImageJ software ( F ). ( n = 3) G Cell viability of HD11 cells treated with OMVs (200 µg/mL) for 12 h was detected by CCK-8 in the absence or presence of 4-PBA (2 mM). ( n = 3) H , I Intracellular survival of HD11 cells infected with WT at MOI = 100 in the absence or presence of 4-PBA (2 mM). Square culture plates showed 1/ 100 of the bacterial load. ( n = 3). n represents three biological replicates; data points indicate independent culture systems. Bar charts display mean ± standard error of the mean (SEM). Data were analyzed using Student’s t -test and two-way ANOVA with Sidak correction. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Rabbit anti-GRP78/BiP polyclonal antibody , Abmart, Shanghai, China , 1:5000.

Techniques: Transmission Assay, Electron Microscopy, Incubation, Labeling, Confocal Microscopy, Imaging, Generated, Software, Western Blot, Expressing, CCK-8 Assay, Infection

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs induce ERS to promote APEC systemic infection . Flowchart of the infection experiment using chicks treated with 4-PBA. B On the sixth day after WT infection of chicks (1 × 10 CFU/chick), the number of bacteria (CFU) was counted in the trachea, lungs, liver, and spleen tissues of the chicks in the absence or presence of 4-PBA (50 mg/kg). Trachea: 95% CI [0.26–0.38]; lungs: 95% CI [0.65–0.78]; liver: 95% CI [0.004–0.13]; spleen: 95% CI [0.08–0.21]. C On the sixth day after WT infection of chicks (1 × 10 9 CFU/chick), HE staining was performed on tracheal, lung, liver, and spleen tissues of the chicks in the absence or presence of 4-PBA (50 mg/kg). Trachea: degeneration and hyperplasia of epithelial mucosal cells (black arrow), diffuse neutrophilic infiltration (red arrow), mucus and inflammatory exudate in the mucosa of the tracheal wall (blue arrow); lung: markedly widened pulmonary septa (black arrow), inflammatory cell infiltration in the pulmonary interstitium (red arrow); liver: hepatocytes show lytic degeneration (black arrow) with inflammatory cell infiltration (red arrow); spleen: marked hyperplasia of lymphoid follicles (black arrow), blurred demarcation between white and red pulp (red arrow). Scale bar, 50 µm or 200 µm. D – F GRP78/BiP immunofluorescence staining was performed on lung and spleen tissues from chicks on day 6 post-infection ( D ) and analyzed using ImageJ software ( E , F ). Scale bar, 50 µm. Data points represent independent cultures; bar charts show mean ± standard error of the mean (SEM). Two-way ANOVA with Sidak correction was performed (* p < 0.05, ** p < 0.01, *** p < 0.001). Effect sizes with 95% confidence intervals are reported in panel ( B ).

Article Snippet: Rabbit anti-GRP78/BiP polyclonal antibody , Abmart, Shanghai, China , 1:5000.

Techniques: Infection, Bacteria, Staining, Immunofluorescence, Software

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: OMVs secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing ERS-mediated autophagy flux blockade . APEC-secreted OMVs, upon uptake by HD11 cells, induce ROS accumulation and Ca 2+ release, triggering ERS and activating UPR pathways, including the PERK, IRE1, and ATF6 signaling branches, leading to the expression of stress-related factors such as GRP78/BiP and CHOP. The sustained activation of ERS inhibits autophagosome degradation and disrupts the acidic environment of lysosomes, thereby preventing autophagosomes from fusing with lysosomes and impairing the phagocytic clearance capacity of macrophages. Collectively, these abnormal conditions facilitate APEC survival within HD11 cells and enable immune evasion, ultimately promoting bacterial dissemination and systemic infection in the host.

Article Snippet: Rabbit anti-GRP78/BiP polyclonal antibody , Abmart, Shanghai, China , 1:5000.

Techniques: Infection, Expressing, Activation Assay