Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Generation of GRP94 conditional KO mice. (A) Identification of GRP94 genotypes of mice used in this study using primers specific for GRP94. GRP94 conditional KO (Pdx1Cre;GRP94flox/flox), conditional heterozygote (HT) (Pdx1Cre;GRP94flox/+), and CTR (Pdx1Cre;GRP94+/+). (B) Identification of Cre recombinase genotype using specific primers. (C) Immunohistochemical staining of GRP94 (green) and Pdx1 (red) in KO and CTR mice at E12.5 (bottom). Scale bar = 50 μm. (D) Immunohistochemical staining of GRP94 (green) and insulin (Ins) (red) in KO and CTR in 4-week-old mice. Nuclei are stained blue. Scale bar = 50 μm. (E) WB analysis of GRP94 protein expression in islets harvested from KO mice. (F) Body weights of CTR and KO mice at 8 weeks of age (n = 4 in each group).

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Deletion of GRP94 in Pdx1+ cells leads to pancreas hypoplasia and reduced β-cell numbers during embryonic development. (A, B) Expression of LacZ (dark blue) in CTR and KO embryos at (A) E10.5 and (B) E18.5. Circled areas in A and B indicate pancreatic tissues, and black arrows in A point to LacZ expression in brain. (C) Hematoxylin and eosin staining of pancreas sections from CTR and KO mice. (D) Quantification of pancreatic area in CTR and KO mice at day 18.5. (E) Micrographs of pancreases and connected intestine from CTR and KO mice. (F) Representative immunohistochemical staining of glucagon (Glu) for α cells (green), insulin (Ins) for β cells (red), and somatostatin (Soma) for δ cells (gray) in pancreases from CTR and KO mice at E14.5, E16.5, and E18.5. Nuclei are stained blue. Scale bar = 50 μm. *P < 0.05, Student t test.

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Deletion of GRP94 reduces proliferation of Pdx1+ cells at E12.5. (A) Representative immunohistochemical staining of pancreases from CTR (n = 4) and KO (n = 5) embryos at E12.5 using anti–E-cadherin (green) and anti-pHH3 (red) antibodies. Nuclei are stained blue. Scale bar = 50 μm. (B) Percentage of pHH3+ cells in Pdx1+ cells. (C) Representative immunohistochemical staining of pancreases from CTR (n = 4) and KO (n = 4) embryos at E18.5 using anti-insulin (Ins; red) and anti-pHH3 (green) antibodies. Nuclei are stained blue. Arrows point to pHH3+Ins+ cells. Scale bar = 50 μm. (D) Percentage of pHH3+ cells in Ins+ cells.

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Immunohistochemical staining, Staining

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Deletion of GRP94 leads to more cell death and ER structural changes in KO mice compared with CTR. (A) Immunohistochemical staining and (B) quantification of TUNEL+ (green) cells among Pdx1+ (red) cells in embryos from CTR (n = 4) and KO (n = 5) mice at E12.5. Nuclei are stained blue. Scale bar = 50 μm. (C) Cleaved caspase-3 (c-Cas-3) staining in pancreatic islets from CTR (n =3) and KO (n = 3) mice at E18.5. GRP94 (gray), glucagon (green), and c-Cas-3 (red). Nuclei are stained blue. White arrows point to c-Cas-3+ cells. Scale bar = 50 μm. (D) Transmission electron microscopy of pancreatic β cells from CTR and KO mice at E16.5. Islets are identified by the presence of insulin-containing granules with a typical electron-dense core, readily seen at higher magnification (right panels). Mt, mitochondria; N, nucleus. Scale bar: left = 2 μm and right = 500 nm. *P < 0.05, Student t test.

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Immunohistochemical staining, Staining, TUNEL Assay, Transmission Assay, Electron Microscopy

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Deletion of GRP94 leads to reduced pancreatic β-cell mass at 4 weeks of age. (A) Pancreas weight/body weight in CTR (n = 4) and KO (n = 4) mice. (B) Images of pancreases from CTR and KO mice. (C) Total islet number from CTR and KO mice. (D) Numbers of small islets (<5000 μm2) in CTR and KO mice. (E) β-Cell mass in CTR and KO mice. (F) α-Cell mass in CTR and KO mice. At least five mice in each group were analyzed. *P < 0.05, Student t test. (G) Representative fluorescent images of pancreatic sections from 4-week-old CTR and KO mice stained for glucagon (green) and insulin (red).

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Staining

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: GRP94 KO mice exhibit impaired glucose tolerance during IPGTT. (A) IPGTT of CTR (n = 5) and KO (n = 5) mice. (B) Areas under the curve (AUC) after glucose injection in CTR and KO mice. (C) Serum insulin levels before (0 minutes) and 15 minutes after glucose injection. *P < 0.05, Student t test. DAPI, 4′6-diamidino-2-phenylindole; Glu, glucagon; Ins, insulin.

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Injection

Journal: Endocrinology

Article Title: GRP94 Is an Essential Regulator of Pancreatic β -Cell Development, Mass, and Function in Male Mice

doi: 10.1210/en.2017-00685

Figure Lengend Snippet: Antibody Table

Article Snippet: Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies against GRP94 (ADI-SPA-850-F; Enzo Life Sciences, Farmingdale, NY), GRP78 (sc-13968; Santa Cruz Biotechnology, Santa Cruz, CA), Glut2 (NBP2-22218; Novus, Littleton, CO), and β -actin (SC-47778; Santa Cruz Biotechnology), and followed by the horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Purification

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: PPARγ Regulates Mitochondrial Structure and Function and Human Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1165/rcmb.2016-0293oc

Figure Lengend Snippet: Figure 6: Hypoxia decreases expression of mitochondrial proteins in HPASMC. HPASMC were treated with hypoxia (HYP, 1% O2) or normoxia (NOR, 21% O2) for 72 h. Protein levels of TFAM (A), GRP75 (B), and VDAC (C) were measured by Western blot analysis. Protein levels of targets were normalized to housekeeping proteins and expressed as mean ± SEM fold-change, relative to NOR. n=8, *p<0.05 vs NOR. Readers may view the uncut gels for Figures 6A, 6B, and 6C in the attached online supplement.

Article Snippet: For HPASMC experiments, membranes were incubated overnight with primary rabbit polyclonal antibodies for GRP75 (1:1000; Cell Signaling Technology Inc., Danvers, MA), VDAC (1:1000; Cell Signaling Technology Inc.), PGC1α (1:500; Calbiochem, San Diego, CA or Cell Signaling Technology Inc.), TFAM (1:1000; Cell Signaling Technology Inc.), and PPARγ (1:500, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Western Blot

Journal: Colloids and surfaces. B, Biointerfaces

Article Title: Breast adenocarcinoma-derived exosomes lower first-contact de-adhesion strength of adenocarcinoma cells to brain endothelial layer.

doi: 10.1016/j.colsurfb.2021.111810

Figure Lengend Snippet: Fig. 2. Panel A: Characterization of MDA-MB-231 cells-derived exosomes based on the absence of Grp94 and on the positive expression of Alix and Hsp70 proteins. Representative western blots are shown of two parallel experiments. Micro vesicles served as negative control (NC). Lys stands for MDA cell lysate. Panel B: Height image of MDA-derived exosomes, immobilized onto glass surface. Image recorded in PBS. Panel C: Size (characterized by particle diameter) distribution of exosomes as measured by Dynamic Light Scattering. Representative data shows three consecutive measurement of the same sample of number 3 on panel D. Panel D: Overall comparison of highest peak positions (size of most abundant particles) from DLS measurements of the used samples.

Article Snippet: Membranes were incubated with primary antibodies against Grp94 (Cell Signaling), Hsp70 (BD Biosciences), Alix (Santa Cruz) overnight at 4 ◦C in TBS-T. After washing the membranes three times for 5 min in TBS-T, the blots were incubated with the secondary antibodies diluted in TBS-T, then washed again three times for 10 min in TBS-T. Immunoreaction was visualized with Clarity Chemiluminescence Substrate (Bio-Rad) in a ChemiDoc MP System (Bio-Rad).

Techniques: Derivative Assay, Expressing, Western Blot, Negative Control, Comparison

Journal: IUBMB life

Article Title: Expression and functional characterization of NPA motif-null aquaporin-1 mutations.

doi: 10.1002/iub.203

Figure Lengend Snippet: Figure 2. Expression of the DNPA mutant and wild type human AQP1 protein. A: Immunoblot analysis of wild type and mutant hAQP1 protein in stably transfected CHO cell clones (CHO/ hAQP1 wild type, CHO/hAQP1DNPA1, CHO/hAQP1D NPA2, CHO/hAQP1DNPA1,2). B: Immunofluorescence showing AQP1 expression in plasma membrane. CHO cells nontransfected with hAQP1 served as control. Double labeling of AQP1DNPA1,2 mutant protein and ER marker GRP94 showing membrane expression pattern of AQP1DNPA1,2. AQP1DNPA1, and AQP1DNPA2 double labeling showed similar results (not shown). Bar: 10 lm.

Article Snippet: In some experiments, dual immunofluorescence labeling was performed using a monoclonal AQP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1:100) and a rabbit polyclonal antibody against ERspecific marker glucose-regulated protein (GRP94) (Boster, Wuhan, China, 1:200) simultaneously and detected with a mixture of Cy3coupled sheep anti-mouse IgG secondary antibody and a FITCcoupled goat anti-rabbit IgG secondary antibody (Sigma).

Techniques: Expressing, Mutagenesis, Western Blot, Stable Transfection, Transfection, Clone Assay, Clinical Proteomics, Membrane, Control, Labeling, Marker

Journal: Cancer Research

Article Title: Enhancement of Cancer Radiation Therapy by Use of Adenovirus-Mediated Secretable Glucose-Regulated Protein 94/gp96 Expression

doi: 10.1158/0008-5472.can-05-0945

Figure Lengend Snippet: Figure 1. Adenovirus-mediated secreted GRP94 gene expression. A, schematic representation of the adenovirus, AdsGRP94GFP, encoding the fusion protein between a modified, secretable GRP94 and GFP. The modification of GRP94 involves the deletion of the COOH-terminal KEDL sequence that anchored the whole protein to the ER. The fusion gene is placed under the control of a constitutively active cytomegalovirus promoter (CMV). B, AdsGRP94GFP-mediated expression of the fusion protein in murine breast tumor 4T1 cells. The cells were infected and irradiated 24 hours later on day 0. Supernatants were collected from the infected cells at different time points after irradiation. Negative control is supernatant collected from control vector AdGFP-infected 4T1 cells. Positive control, cellular lysates from AdsGRP94GFP-infected Ad293 cells. The antibody used was against GRP94; it was purchased from StressGen.

Article Snippet: The antibody used was against GRP94; it was purchased from StressGen. www.aacrjournals.org 9127 Cancer Res 2005; 65: (20).

Techniques: Gene Expression, Modification, Sequencing, Control, Expressing, Infection, Irradiation, Negative Control, Plasmid Preparation, Positive Control

Journal: Cancer Research

Article Title: Enhancement of Cancer Radiation Therapy by Use of Adenovirus-Mediated Secretable Glucose-Regulated Protein 94/gp96 Expression

doi: 10.1158/0008-5472.can-05-0945

Figure Lengend Snippet: Figure 4. Efficacy of combined radiation and secreted GRP94-mediated immunotherapy in established murine 4T1 tumors. A, treatment scheme for the combined therapies. B, tumor growth delay as a result of the combined treatment. For this graph, day 0 is the day when radiation treatment started. Points, mean of 10 to 12 mice; bars, SE. GRP, vaccination with AdsGRP94GFP-infected 4T1 cells and i.t. injection of the AdsGRP94GFP (1 108 pfu per injection). GFP, vaccination with AdGFP-infected 4T1 cells and i.t. injection of AdGFP (1 108 pfu per injection). C, Kaplan-Meier plots of host BALB/c mice survival as a result of the combined treatment. The group designations are the same as in (B). D, kinetics of de novo metastases in mice that have undergone combined radiation- and sGRP94-mediated immunotherapy. 4T1-Luc cells, which had been stably transduced with the firefly luciferase gene, were injected i.v. into mice bearing established tumors that had undergone various treatments. The growth of tumors from these cells was then followed with the aid of a very sensitive Xenogen IVIS bioluminescence detection system. Top, Kaplan-Meier plots of metastasis-free rate in the 4T1-Luc-injected mice. Points, value from five to seven mice. The treatment group designations are the same as those in (B). Bottom left, representative photographs of luciferase signal as a result of 4T1-Luc growth. Bottom right, confirmation from dissected lungs of tumor growth from the mice shown in left. Abbreviations: TF, tumor free; TG, tumor growth; RT, radiation therapy.

Article Snippet: The antibody used was against GRP94; it was purchased from StressGen. www.aacrjournals.org 9127 Cancer Res 2005; 65: (20).

Techniques: Infection, Injection, Stable Transfection, Transduction, Luciferase