Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

doi: 10.4049/jimmunol.1001822

Figure Lengend Snippet: (A). Representative EM images of naïve Atg7-deficient and wildtype CD4+ T cells (left two panels) and quantification of membrane structures in EM images (right panel figure). Each cell was divided into 12 equal sections based on the face of a clock. Membrane scores were defined as the number of sections per cell containing membrane structure(s). The bar figure are mean and SD (*n=50, p=3.23×10-7 for CD4 cells and **n=45, p=2.63×10-19 for CD8 cells). (B). Calnexin protein expression in purified total T cells from Atg7f/fLck-Cre and Atg7f/f mice was analyzed by Western blot. Numbers represent the ratios of intensity of calnexin bands to actin bands (upper panel). This Western blot analysis was repeated three times using samples from three different pairs of wildtype and Atg7f/fLck-Cre mice. The normalized intensities of three independent experiments are showed in the lower panel figure (mean±SD, *p=0.04). (C). Expression of ER stress markers in autophagy-deficient T cells. Cell lysates of purified total T cell from Atg7f/f or Atg7f/fLck-Cre mice splenocytes were prepared. The expression levels of PDI, Grp78, and Grp94 were analyzed by Western blot. Numbers represent the ratios of the intensity of target molecule band to the intensity of actin band. This Western blot was repeated twice using three pair of wildtype and Atg7f/fLck-Cre mice.

Article Snippet: Anti-Grp78, anti-Grp94, and anti-PDI antibodies are from Assay Designs (Ann Arbor, Michigan).

Techniques: Expressing, Purification, Western Blot

Journal: PLoS ONE

Article Title: Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

doi: 10.1371/journal.pone.0132283

Figure Lengend Snippet: Mass spectrometric identification of CREC interacting proteins.

Article Snippet: Recombinant GST-grp75 (AAH00478.1) was obtained from Abnova (Taipei, Taiwan).

Techniques:

Journal: PLoS ONE

Article Title: Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

doi: 10.1371/journal.pone.0132283

Figure Lengend Snippet: (A) 35 S-labelled MRC-5 V2 proteins were separated by 2D-PAGE, blotted onto a membrane and subjected to autoradiography. The autoradiogram is shown. (B) The membrane was used for Western blotting by incubating with anti-reticulocalbin antiserum. The antibody showed a strong reaction against two reticulocalbin spots (white arrows) and a slight cross-reaction against an unknown protein (small black arrow). (C) 35 S-labelled MRC-5 V2 proteins were immunoprecipitated with anti-reticulocalbin antiserum mixed with unlabelled proteins and separated by 2D-PAGE with subsequent silver staining. (D) Autoradiogram of 35 S-labelled, immunoprecipitated proteins. Two reticulocalbin spots were immunoprecipitated (white arrows) as well as protein 2 and 3. Proteins 2 and 3 were identified as grp75 by mass spectrometry (see text and ). The cross-reacting protein was not immunoprecipitated. (E) No proteins were immunoprecipitated without the presence of anti-reticulocalbin antiserum.

Article Snippet: Recombinant GST-grp75 (AAH00478.1) was obtained from Abnova (Taipei, Taiwan).

Techniques: Membrane, Autoradiography, Western Blot, Immunoprecipitation, Silver Staining, Mass Spectrometry

Journal: PLoS ONE

Article Title: Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

doi: 10.1371/journal.pone.0132283

Figure Lengend Snippet: (A) Calumenin was immobilised to the chip and various concentrations of GST-grp75 (AAH00478.1) were applied at indicated concentrations in buffer with EGTA (10 mM Hepes, 150 mM NaCl, 5 mM CaCl 2 , 1 mM EGTA and 0.005% Tween 20 pH 7.4). The dissociation constant was estimated to be approx. 7 nM. (B) Various concentrations of GST-grp75 (AAH00478.1) were applied at indicated concentrations in buffer with EGTA (10 mM Hepes, 150 mM NaCl, 5 mM CaCl 2 , 1 mM EGTA and 0.005% Tween 20, pH 7.4). The dissociation constant was estimated to be approx. 3 nM.

Article Snippet: Recombinant GST-grp75 (AAH00478.1) was obtained from Abnova (Taipei, Taiwan).

Techniques:

Journal: PLoS ONE

Article Title: Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

doi: 10.1371/journal.pone.0132283

Figure Lengend Snippet: (A) MRC-5 V2 cells were transfected with fibulin-1C and stained for reticulocalbin (red fluorescence) and (B) fibulin-1C (green fluorescence). (C) The merged picture shows colocalization of reticulocalbin and fibulin-1C (yellow fluorescence). (D) MRC-5 V2 cells were transfected with fibulin-1C and stained for calumenin (red fluorescence) and (E) fibulin-1C (green fluorescence). (F) The merged picture shows colocalization of calumenin and fibulin-1C (yellow fluorescence). (G) MRC-5 V2 cells stained for reticulocalbin (red fluorescence) and (H) grp75 (green fluorescence). (I) The merged picture shows colocalization of calumenin and grp75 (yellow fluorescence). Each of the merged pictures were made with the same magnification as each of the individual stainings.

Article Snippet: Recombinant GST-grp75 (AAH00478.1) was obtained from Abnova (Taipei, Taiwan).

Techniques: Transfection, Staining, Fluorescence

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 is an interactive partner of cholix. Polarized monolayers of Caco-2 cells were exposed apically to nontoxic full-length Chx (ntChx) anchored through a C-terminal hexa-histidine sequence to 100 nm diameter magnetic beads. After 15 min, cell membranes were isolated and separated into non-magnetic and magnetic fractions. a, 1-D SDS-PAGE gel of non-magnetic and magnetic captured membrane fractions. b, 2-D gel separation of proteins present in non-magnetic and magnetic capture membrane fractions with location of spot 16 identified by mass spectrometry as GRP75. c, Pull-down (PD) using ntChx probed with antibodies to either GRP75 or human growth hormone (hGH). d, Immunoprecipitation of Caco-2 cell lysate using ntChx probed with anti-GRP75 or control antibodies. e, 2-D Western blot of magnetic capture membrane fraction probed with anti-GRP75. f, ELISA-based, neutral pH, format where bound ntChx or the control protein bovine serum albumin (BSA) was used to capture GRP75, heat shock protein 60 (HSP60), glucose-regulated protein 78 (GRP78), or HSP90.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Sequencing, Magnetic Beads, Isolation, SDS Page, Membrane, Mass Spectrometry, Immunoprecipitation, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 and perlecan are functionally involved in cholix transcytosis. Expression of three proteins identified by ntChx-magnetic bead capture were suppressed using a CRISPR/cas 9-mediated knockdown protocol that targeted either GRP75, HSPG2 , or KRT8 in Caco-2 cells. Confluent monolayers of the resulting cell lines having decreased levels of a, GRP75, b, perlecan, or c, cytokeratin 8 (CT8) were evaluated for their capacity for Chx-mediated transcytosis using Chx266-hGH and for the nonspecific movement of the control protein hGH over a 60 min time period. d, Transcytosis of Chx266-hGH across confluent monolayers of parent Caco-2 cells was evaluated following a simultaneous apical application of a monoclonal antibody against GRP75 or perlecan, with an isotype antibody recognizing interleukin (IL)-10 serving as a control. Densitometry measurements of individual Western blot bands are shown for graphical comparison.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Expressing, CRISPR, Knockdown, Control, Western Blot, Comparison

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 associates with cholix in apical endosomes. Immunofluorescence microscopy was performed on rat jejunum over a time course following intraluminal injection (ILI) of AMT-101. Co-localization of the human interleukin (IL)-10 element of AMT-101 with TMEM132A (a, c) or GRP75 (b, d) at 1- or 5-min post ILI. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Relationship of GRP75 to GRP78 and TMEM132A. Immunofluorescence microscopy was performed on rat jejunum over a time course following intraluminal injection (ILI) of Chx266-hGH. Co-localization events detecting the human growth hormone (hGH) element of Chx266-hGH with a, TMEM132A or b, GRP78 compared to the co-localization of c, TMEM132A and GRP78 at 5 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 does not associate with cholix in basal vesicular structures. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with early endosomal antigen 1 (EEA1; a, c) or GRP75 (b, d) at 5- or 15-min post ILI. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Cholix trafficking involves LMAN1-positive compartments. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with a, early endosomal antigen 1 (EEA1) or b, LMAN1 at 5 min post ILI. c, Co-localization of EEA1 and LMAN1 at 5 min post ILI. Co-localization of hGH with d, GRP75 or e, LMAN1 at 15 min post ILI of Chx266-hGH. f, Co-localization of GRP75 and LMAN1 at 15 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: GRP75 intersects with furin differently than with LMAN1. Immunofluorescence microscopy was performed on rat jejunum after intraluminal injection (ILI) of Chx266-hGH. The human growth hormone (hGH) element of Chx266-hGH was co-localized with a, furin or b, GRP75 at 15 min post ILI. c, Co-localization of furin and GRP75 at 15 min post ILI. Co-localization of hGH with d, furin or e, LMAN1 at 15 min post ILI of Chx266-hGH. f, Co-localization of furin and LMAN1 at 15 min post ILI of Chx266-hGH. Apical (luminal) epithelial membrane (open arrow); basal epithelial cell surface-basement membrane demarcation (dashed line); lamina propria ( l-p ); goblet cell (g). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue).

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Immunofluorescence, Microscopy, Injection, Membrane, Staining

Journal: Tissue Barriers

Article Title: GRP75 as a functional element of cholix transcytosis

doi: 10.1080/21688370.2022.2039003

Figure Lengend Snippet: Diagram of events associated with cholix apical to basal transcytosis with interaction partners. Highlighted steps are explained in the text with greater detail. (1) Cholix enters at the apical plasma membrane in the microvilli area where it interacts with TMEM132A and furin. (2) Cholix enters an early endosomal compartment where it traffics preferentially to a late endosome rather than a recycling pathway. (3) Instead of continuing along the lysosomal degradation pathway, cholix interacts with GRP75 and moves to a sorting endosome. (4) Reorganization of LMAN1 from the endoplasmic reticulum intermediate Golgi complex (ERGIC) to apical sorting vesicles. (5) In this location, LMAN1 can intersect with cholix delivered to this site with furin and GRP75. (6) LMAN1 and furin appear to return with cholix to a supranuclear region of the cell consistent with location of the ERGIC. (7) Reorganization of LMAN1 from the ERGIC to the basal vesicular compartment provides a route for cholix to this region of the cell. (8) Cholix in complex with LMAN1 enters sorting endosomes in the basal vesicular compartment. (9) Within basal sorting endosomes, cholix/LMAN1 intersects with perlecan in a recycling endosome, which delivers it to the basal plasma membrane, resulting in cholix exocytosis and completion of the apical to basal transcytosis process. (10) Perlecan recycles to the basal sorting endosome where it can engage cholix trafficked to this site with LMAN1. (11) Unlike in the apical vesicular compartment, GRP75 and furin present in the basal vesicular compartment are not associated with cholix in this region of enterocytes.

Article Snippet: Western blotting was performed using rabbit anti-human GRP75 as primary antibody followed by using Cy3-labeled Donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 1;2500).

Techniques: Clinical Proteomics, Membrane

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1 signaling regulates ER calcium levels. Schematic (I) Timeline of experiments assessing changes in ER calcium levels. Schematic (II) Timeline of experiments, in cells treated with siRNA before assessing changes in ER calcium levels. (A) Fluo4 fluorescence in HEK cells expressing NIC1-RFP ( ) or RFP ( ), at time-t (F t ) relative to onset (F 0 ) measured in calcium free medium at baseline and in response to 2 μM TG. (B) YFP/CFP ratio in HEK cells co-transfected with D1ER and NIC1-RFP ( ) or, Bcl-xL-RFP ( ) or, RFP ( ) and cultured for 36 h before imaging. (C) Schematic (not to scale) depicting - IP3R3, Grp75, VDAC1 and MCU- in the context of ER and mitochondria. (D–F) Ratio of YFP/CFP fluorescence in HEK cells, imaged 36 h after co-transfection with D1ER and NIC1-RFP ( ) or RFP ( ), in cells pre-treated with siRNA to IP3R3 (D-ii) or VDAC1 (E-ii) or MCU (F-ii) or scrambled control ( D-F-i , NIC1-RFP ( ) and RFP ( ). (D–F iii) Percent mRNA levels of the genes as shown in panels, in cells treated with siRNA to IP3R3 ( D , N=3) or VDAC1 ( E , N=2) or MCU ( F , N=3) and scrambled control. Data plotted as mean ± SD of the indicated number of cells in 6-12 fields across three (A, D, F) or two (B, D) independent experiments. *** indicates significant difference at all time points with p-values ≤0.001 and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Fluorescence, Expressing, Transfection, Cell Culture, Imaging, Cotransfection, Control

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: NIC1-mediated Treg survival requires IP3Rs, Grp75 and MCU. (A) Percent apoptotic nuclei in activated WT Tregs cultured without IL-2 for 24 h with vehicle control (UT) or, 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (B, C) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or NIC1-NES (B) or pMIG or Bcl-xL (C), and cultured without IL-2 for 24 h with vehicle control, or 5 μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (D) Basal and maximum OCR, computed as described in methods, in Notch1 -/- Tregs transduced with pBABE or NIC1-NES. (E) Immunoblot of whole cell lysates prepared from activated WT Tregs cells cultured with or without 10 μM GSI-X or 10 μM MKT-077 for 8 h and the samples run in duplicate. Membrane was probed either for (p)PDH, Notch1 and Tubulin (E-i) or PDH, Notch1 and Tubulin (E-ii) . The immunoblot is representative of two independent experiments. Data are mean ± SD of three independent experiments (A–C) and readings in 4 wells from two independent experiments (D). ** indicates significant difference with p-value ≤0.01 and ns, not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Cell Culture, Control, Transduction, Western Blot, Membrane

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Notch1 activity regulates Grp75 protein levels. (A) Immunoblots of whole cell lysates prepared from activated Notch1 +/+ (Cre-ve) and Notch1 -/- (Cre+ve; Cd4-Cre::Notch1 lox/lox ) Tregs, run in duplicate. Membranes were sequentially probed for Grp75, VADC1 (*VDAC1 band), and Actin (A-i) or Notch1, MCU and Actin (A-ii). Mean ± SD values below are the densitometry analysis of Grp75, VDAC1 and MCU relative to Actin. (B) Representative Z-projected confocal images of Notch1 +/+ and Notch1 -/- Tregs immune-stained with an antibody to Grp75 (green). scale bar: 5 μm. Images are representative of 106 Notch1 +/+ Tregs and 82 Notch1 -/- Tregs. (C) Relative transcript levels of indicated genes in Notch1 +/+ and Notch1 -/- activated Tregs. (D) Immunoblots of cell lysates from Notch1 -/- Tregs transduced with pBABE or NIC1-NES probed for Notch1, Grp75 and Actin. Mean ± SD values below are the densitometry analysis of Grp75 relative to Actin. (E) Percent apoptotic nuclei in Notch1 -/- Tregs transduced with pBABE or Grp75, cultured without IL-2 for 24 h with 5μM Xestospongin C or, 10 μM MKT-077 or, 10 μM Ru360. (F) Basal and maximum OCR in Notch1 -/- Tregs transduced with pBABE or Grp75. Control (pBABE) condition in panels E and F are common Figures 5B, D as these were tested in the same experiment, as described in methods . (G, H) Cell lysates of WT activated Tregs cultured for 6 h without IL-2 were subject to immunoprecipitation using an antibody to Notch1 (G) or Grp75 (H) or, IgG (Isotype control), and associated proteins analyzed by western blotting for Grp75, VDAC1 (*shows VDAC1), Notch1, Vps34, Actin and IgG (Isotype control). Immunoblots are representative of three independent experiments (A) or, two independent experiments (D, G, H) . Data show the mean ± SD of three independent experiments (C, E) and readings in 4 wells from two independent experiments (F). * and ** indicates significant difference with p-value ≤0.05 and ≤0.01 respectively, and ns: not significant, examined using unpaired student’s t-test.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques: Activity Assay, Western Blot, Staining, Transduction, Cell Culture, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Notch1 Modulation of Cellular Calcium Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in T-Regulatory Cells

doi: 10.3389/fimmu.2022.832159

Figure Lengend Snippet: Summary of key outcomes. Non-nuclear localized NIC1 interacts with Grp75 and VDAC1 in a complex that modulates calcium homeostasis in mitochondria with consequences to cellular metabolism and survival. The schematic is not to scale.

Article Snippet: Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector.

Techniques:

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: Cells

Article Title: Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate

doi: 10.3390/cells13060523

Figure Lengend Snippet: List of all the used antibodies.

Article Snippet: GRP75 , 75 , 1:1000 , - , Cell Signaling Technology, Danvers, MA, USA #2816.

Techniques: Molecular Weight, Plasmid Preparation

Journal: Cells

Article Title: Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate

doi: 10.3390/cells13060523

Figure Lengend Snippet: Analysis of calcium signaling ( A ) and ER stress ( B ) in control and D-Asp-treated rats. ( A ) Western blot analysis of VDAC (32 kDa) and GRP75 (75 kDa) protein levels in the testes of animals treated with D-Asp for 15 days compared to controls. ( B ) Western blot analysis of GRP78 (78 kDa) protein levels in rat testes of D-Asp group compared to controls. The expression levels of VDAC, GRP75, and GRP78 were quantified using ImageJ and normalized to β-actin (42 kDa). All values are expressed as the mean ± SD of 5 animals in each group. D-Asp vs. Control (C): * p < 0.05.

Article Snippet: GRP75 , 75 , 1:1000 , - , Cell Signaling Technology, Danvers, MA, USA #2816.

Techniques: Control, Western Blot, Expressing