Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: 2D side-by-side comparison of the 2-DE Western blot images of HSP27, PRDX2, GRP78 and GRP75 in cholesteatoma tissues and retroauricular skin run with pI 4–7 and pI 3–10, respectively. Images A , B , C and D are for HSP27. Images E , F , G and H are for PRDX2. Images I , J , K and L are for GRP78. Images M , N , O and P are for GRP75.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: Validation of HSP27, GRP75, GRP78, GRP94 and PRDX2 by Western blotting analysis and RT-PCR. The tissues of cholesteatoma and retroauricular skin were collected from six individual patients. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Biomarker Discovery, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: Available sera from 39 patients were sampled 7 days before onset (D-7), the day of acute GvHD onset, and 7 days and 14 days after diagnosis (D+7 and D+14, respectively). Gp96 was quantified by ELISA. (A) All acute GvHD patients included. It is worth noting that, for some patients, data at one specific time point may be missing. Statistical analysis performed using a 1-way ANOVA test (Dunn’s multiple comparisons test). *P < 0.05. (B) Patients (n = 17) with grade II (n = 10) vs. III–IV intestinal (GI) GvHD (n = 7). Statistical analysis performed using a 2-way ANOVA test (Tukey’s multiple comparisons test). **P < 0.01, ****P < 0.0001. (C) Patients presenting isolated skin GvHD (n = 18). (D) Non-GvHD patients (n = 7). Gp96 levels were measured 3 weeks after hematopoietic stem cell transplantation (median: D21, most often the onset of GvHD) and 1 week later (median: D28).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Isolation, Transplantation Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Left panel: mouse model of acute GvHD. Syn: syngeneic graft (no GvHD developed). BM: allogeneic graft without splenocytes (no GvHD). Allo: allogeneic graft with splenocytes (acute GvHD). Right panel: survival of the mice belonging to the different groups. (B) Western blot analysis of Gp96 and HSP90 in sera at day 7 after transplantation. Recombinant proteins were run as a control. (C) Western blot analysis of Gp96 expression at the indicated days after transplantation in sera of mice developing GvHD (Allo) or not (Syn). The lanes were run on the same gel but were noncontiguous. (D) Proportion of splenic macrophages (according to F4/80 staining) expressing Gp96 at the surface was determined by FACS analysis 7 days after hematopoietic stem cell transplantation. Statistical analysis performed using a 1-way ANOVA test. ****P < 0.0001. A representative experiment is shown (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Transplantation Assay, Recombinant, Control, Expressing, Staining

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Western blot analysis of Gp96. (B) Coomassie blue coloration of proteins coimmunoprecipitated with Gp96 (bottom panel) in mice developing GvHD (Allo) or not (Syn) 7 days after hematopoietic stem cell transplantation. Ctl, mice with no transplantation. The proteins indicated were determined by mass spectrometry. One representative experiment is shown (n = 2). (C) Protein-protein interaction data between Gp96 or HSP60 immobilized onto the biosensor and C3 (left panel) or C3b (right panel) as analytes.

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Transplantation Assay, Mass Spectrometry

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Linear structure of C3 (β-chain in yellow, α-chain in blue) and of the tested complement C3 fragments (see also Supplemental Figure 2). (B) Yeast two-hybrid assay between the C3 fragments described in A and full-length Gp96. A representative picture is shown. (C) Linear structure of Gp96 and the analyzed deletion mutants. ABD, ATP-binding domain; Ca2+, charged linker domain; PBD, peptide-binding domain. (D) Two-hybrid assay between complement C3 749–1,303 amino acid fragment and Gp96 deletion mutants. A representative image is shown (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Y2H Assay, Binding Assay, Two Hybrid Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: The N-terminus of Gp96 is indicated. Each structure is represented by a transparent surface superposed to the ribbon diagram of the protein backbone. The color code is the following: red for the C3 fragment (749–955), blue for the Gp96 and pink for the C3 complementary structure (1–748; 956–1641). (A and B) Two views of the Gp96-C3 fragment model. (C and D) Two views of the Gp96 full-length C3 model. This figure was prepared with PyMOL (http://www.pymol.org). N = N-terminal domain.

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques:

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Gp96 inhibits C3b cleavage by factors I and H. Upper panel: Western blot analysis of the α’-chain (C3b) and the α2-chain (iC3b and C3c) both labeled with anti-C3c antibody, and of the α1-dg-chain (iC3b) labeled with anti-C3d antibody, after C3b incubation (0.3 μM) with normal human serum, supplemented with factors I and H with or without purified human Gp96 (1.2 μM). Lower panel: densitometry quantification. Cleavage without Gp96 is rationalized to 1. Statistical analysis performed using a 2-tailed Mann-Whitney U test. *P < 0.05; **P < 0.01 (n = 4 or 5). (B) Gp96 effect on opsonophagocytosis. Flow cytometry analysis of phagocytosis by human purified macrophages of Alexa Fluor 488–conjugated (AF 488–conjugated) E. coli bioparticles after opsonization by healthy human serum with or without Gp96 (1.5 mM). A representative image of AF 488 fluorescence in living cells is shown: the filled gray curve represents opsonization without serum, gray line with serum alone, black line with serum and Gp96, and dotted black line with serum and “BSA in buffer.” Histograms of mean fluorescence intensity ± SEM are shown. Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3). (C) Gp96 effect on opsonization. AF 488–E. coli bioparticles were incubated with serum with or without Gp96 (1.5 mM). C3 and C4 were determined by Western blot in supernatants after deesterification of proteins tagged on bioparticles. One representative experiment is shown (n = 3). (D) Microscopy on AF 488–bioparticles after C3 AF568 staining. Right panel: C3 bioparticle quantification (median of fluorescence of AF 568 staining ± SEM). Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3). (E and F) Gp96 effect on complement activation pathways. (E) Antibody-coated sheep erythrocytes (for classical pathway) (n = 6) and (F) rabbit erythrocytes hemolysis (for alternative pathway) (n = 4), in presence of Gp96 or controls. Percent of hemolysis are shown. Statistical analysis performed using a 2-tailed Mann-Whitney U test. *P < 0.05; **P < 0.01 (E: n = 6; F: n = 4).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Labeling, Incubation, Purification, MANN-WHITNEY, Flow Cytometry, Fluorescence, One-tailed Test, Microscopy, Staining, Activation Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Left, schematic representation of the model of GvHD used with a C3–/– recipient: FvB/N → WT or C3–/– C57BL/6. Right, Western blot analysis of C3 and Gp96 in day 7 sera of WT or C3–/– mice, developing GvHD (Allo) or not (Syn and BM). (B) Top, schematic representation of the mice model with a C3–/– donor: WT or C3–/– C57BL/6 → BALB/c. Lower left panel, Western blot analysis of C3 and Gp96 in day 7 sera of mice receiving a WT or a C3–/– graft, developing GvHD (Allo) or not (Syn and BM). Samples from 7 animals per group were pooled. Lower right panel, immunoprecipitation of Gp96 in the sera from the different described animals groups was followed by complement C3 immunoblotting. One representative experiment out of 3 performed is shown. (C) Survival of the mice belonging to the different groups described in B, (n = 4) from 2 independent experiments. (D) Percentage of activated, splenic T cells (CD3+CD69+) 7 days after hematopoietic stem cell transplantation in mice receiving either a WT or a C3–/– graft, developing GvHD (Allo) or not (Syn and BM). Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Immunoprecipitation, Transplantation Assay, One-tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

doi: 10.1083/jcb.201904004

Figure Lengend Snippet: Hyperglycosylation of GRP94. (A) The domain structure and location of glycosylation sites in GRP94. (B) The Δlog 2 values for GRP94 sites in STT3A −/− and STT3B −/− cells. Error bars designate SDs ( n = 3–8) unless denoted by an asterisk ( n = 2). Not quantifiable (NQ) and not detected (ND) report results for the STT3B −/− cells. (C) Protein (50 µg) in lysates from WT and mutant cells was analyzed by protein immunoblotting using antisera to GRP94 and the α-subunit of ATP synthase. EH designates digestion with EH. (D) EH digestion time course of pulse-labeled GRP94. (E) Pulse labeling of GRP94 in similar numbers of WT and mutant cells. The right hand lane is a lighter version of the preceding lane. (F) Pulse labeling (5 min) of GRP94 in STT3A −/− cells followed by the indicated chase times. (G) Pulse labeling of human GRP94-DDK-His and GRP94-DDK-His N217Q mutant in WT and mutant cells. (H) Pulse labeling of GRP94 in WT or STT3A −/− cells treated with the following compounds: DTT (200 µm), tunicamycin (Tn, 0.6 µM), thapsigargin (Tg, 0.1 µM), and NGI-1 (10 µM). (I) Pulse labeling of GRP94 in control fibroblasts (C-1 and C-2), STT3A-CDG fibroblasts, and WT and STT3A −/− cells. The EH-digested sample is from the STT3A −/− cells and is equivalent to 10% of the undigested sample.

Article Snippet: Mouse monoclonal antibodies to GRP94 (MAB7606; R&D Systems), GLUT1 (ab40084; Abcam), the α-subunit of the F o F 1 ATPase (612516; BD Biosciences), and the DDK epitope tag (F3165 anti-FLAG M2; Sigma-Aldrich) were obtained from commercial sources.

Techniques: Glycoproteomics, Mutagenesis, Western Blot, Labeling, Control

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc increase S protein specific IgG responses in vivo. a) C57Bl6 mice were vaccinated at day 0 and 14 with different concentrations of cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S and OX40L-Fc or with AD100 or PBS (controls). b) Mice were vaccinated at day 0 and 14 with 1μg/ml of ZVX-55 and ZVX-60 or with AD100 and PBS (controls). Serum was collected 5 days after last vaccination, and S protein specific IgG response was analyzed by ELISA. c) Mice were vaccinated at day 0 and 14 with 1 μg/ml ZVX-55 or ZVX-60 and S protein specific IgG response in serum was analyzed at day 5, 14 and 19. Data represent 3 independent biological replicates per group and mean ± standard error. To compare control (ZVX55) with experimental (ZVX60) group (alpha level of 0.05) unpaired t-test (two-tailed) was applied, *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce B cells responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD19, IgM, CD21, CD23, CD49, CD93. Frequency of CD19+IgM+ (activated B cells) and CD21+CD23- (marginal zone, MZ), CD21+CD23+ (follicular, FO) and CD21-CD23- (double negative or ABC cells) CD19+IgM+ cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of activated B cells (CD19+IgM+) within total CD45+ T cells and frequency of FO (CD21+CD23+) within CD19+IgM+ cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce T follicular helper (TFH) cell responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD4, PD1 and CXCR5. Frequency of PD1+CXCR5+ (TFH cells) within CD4+ T cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of TFH cells (PD1+CXCR5+) within total CD4+ T cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Characteristics of the cell line expressing gp96-Ig, SARS-CoV-2 Spike (S) protein and OX40L-Fc. Cell line (AD100) was transfected with plasmids encoding a) gp96-Ig and full length protein S and b) gp96-Ig, full length protein S and OX40L-Fc. c) Secreted gp96-Ig was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified gp96-Ig was used as standard d) Secreted OX40L-Fc was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified OX40L-Fc was used as standard e) SARS-CoV2 protein S expression was analyzed by immunofluorescence f) SARS-CoV2 protein S expression in supernatant was measured by ELISA. One million cells were plated in 1 ml for 48 h and purified SARS-CoV2 protein S was used as standard.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Purification, Immunofluorescence

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Enhancement of S1- specific CD8+ T cell responses by Gp96-Ig-S-OX40L-Fc in the spleen, lung tissue, and BAL . a) 5 days after the vaccination of HLA-A2 transgenic mice (n=3-5) with the ZVX-55, ZVX-60 vaccine cells (1ug secreted gp96-Ig) or AD100 or PBS (controls), splenocytes (SPL), lung cells and bronchioalveolar lavage (BAL) cells were isolated from vaccinated and control mice (PBS). Cells were stained with HLA-A2 02-01 pentamer containing YLQPRTFLL peptides, followed by surface staining for CD45, CD3, CD4, CD8, CD69, CXCR6. Bar graphs represent percentage of the pentamer positive cells within CD8+ T cells. b) 5 days after the vaccination of C57Bl6 mice (n=3), splenocytes and lung cells were isolated from vaccinated and control mice (PBS and AD100) and in vitro restimulated with S1 and S2 overlapping peptides in the presence of protein transport inhibitor, brefeldin A for the last 5 hours of culture. After 20 hours of culture, intracellular cytokine (IFNg, TNFa and IL-2) staining was preformed to quantify protein S-specific CD8+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. Data represent at least 2 technical replicates with 3–5 independent biologic replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Transgenic Assay, Isolation, Staining, In Vitro, Expressing

Journal: Molecular Metabolism

Article Title: TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion

doi: 10.1016/j.molmet.2018.01.016

Figure Lengend Snippet: TALK-1 channels are expressed in mouse and human δ-cells. (A) Mouse pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 mice). (B) Mouse pancreas section stained for TALK-1 (green), ER (GRP94, red), and SST (cyan). (C) Human pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 pancreata). (D) Human pancreas section stained for TALK-1 (green), ER (GRP94, red), and somatostatin (E) K2P currents recorded from WT and TALK-1 KO δ-cells ( N = 3 mice per genotype). (F) K2P currents recorded from human δ-cells expressing TALK-1 DN or control mCherry. ( N = 3 islet preparations); * P < 0.05, ** P < 0.005.

Article Snippet: Sections were stained using primary antibodies against somatostatin (Santa Cruz Biotechnology sc-7819: 1:250), TALK-1 (Novus Biologicals #NBP1-83071; 1:175) or TALK-1a (Antibody Verify AAS72353C; 1:250), glucagon (Proteintech #15954-I-AP: 1:500), and the endoplasmic reticulum marker GRP94 (Novus Biologicals #NB300-619; 1:100); secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (Jackson Immunoresearch #711-546-152; 1:300), DyLight 650-conjugated donkey anti-goat (Thermo Fisher #SA5-10089; 1:250), and Cy3-conjugated donkey anti-mouse (Jackson Immunoresearch #715-166-150; 1:500).

Techniques: Staining, Expressing, Control