Journal: Journal of Immunology Research

Article Title: Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

doi: 10.1155/2016/6078473

Figure Lengend Snippet: Overexpression of mHSF1 does not impair the phenotype and function of immature and mature DCs. Immature DCs were transduced with Ad5Luc1 or Ad5mHSF1 at 500 TCID 50 /cell or Mock-treated and were either left immature (“iDC”) or were matured (“mDC”) with a maturation cytokine cocktail (IL-1 β , IL-6, TNF- α , and PGE 2 ). (a) Western Blot analyses of cell lysates harvested 24 h after transduction on HSF1, mHSF1, Hsp40, Hsp70A, Hsp70B′, Hsp90, Grp94, and beta-actin protein expression. One representative experiment out of three is shown. (b, c) Flow cytometric analyses of iDCs and mDCs on CD25, CD80, CD83, CD86, HLA-ABC, and HLA-DR (b) as well as PI (c) 24 h and 48 h after transduction. Data are mean ± SEM of four independent experiments with DCs derived from different donors. (d) Cell culture supernatants derived from experiments shown in (b and c) were analyzed by cytometric bead array for the content of IL-1 β , IL-6, IL-8, IL-10, IL-12p70, and TNF- α . The red line in (d) indicates background levels of respective cytokines derived from the cytokines present in the maturation cocktail. Data are mean ± SEM of three independent experiments. (e) Twenty-four hours after adenoviral transduction accompanied by maturation of DCs, cells were either transfected with wild-type MelanA RNA (left panel) or loaded with MelanA analogue peptide (right panel). Antigen-loaded DCs were cocultured with MACS-sorted autologous CD8 + T cells for seven days. Induction of antigen-specific CTLs was determined by HLA-A2-MelanA/iTag MHC class I-tetramer and anti-CD8-PC7 staining using a FC500 cytofluorometer. Data are mean ± SEM of three independent experiments with different donors. (b–e) ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and n.s.: not significant ( P > 0.05); bars without annotation are not significant ( P > 0.05), one-way, (e) or two-way ANOVA (b–d) with Bonferroni's Multiple Comparison post hoc test.

Article Snippet: Membranes were incubated with primary antibodies (Abs) diluted 1 : 1000–1 : 100 against HSF1, Hsp40, Hsp70A (C92F3A-5), Hsp70B′, Hsp90 (AC-88), Grp94 (9G10) all from Stressgen (distributed by Biomol, Hamburg, Germany), BclxL (2H12; Calbiochem/Merck-Millipore, Darmstadt, Germany), MelanA (A103; Sigma-Aldrich), or beta -actin (AC-74; Sigma-Aldrich), followed by HRP-conjugated horse anti-mouse IgG, goat anti-rabbit IgG, or goat anti-rat IgG Abs (all Cell Signaling Technology, UK) diluted 1 : 10000–1 : 2000.

Techniques: Over Expression, Transduction, Western Blot, Expressing, Derivative Assay, Cell Culture, Transfection, Staining

Journal: Brazilian Journal of Microbiology

Article Title: Identification of GRP75 as a novel PreS1 binding protein using a proteomics strategy

doi: 10.1590/S1517-838220100002000036

Figure Lengend Snippet: 2-DE analysis of GST-PreS1 binding protein. GST pull down using GST (A), GST-PreS1 (B) with binding buffer, GST (C) and GST-PreS1 (D) with HepG2 cell lysate. IEF on Immobiline DryStrips (pH range 3-10) followed by SDS-PAGE and silver staining. Protein spots 1 and 2 (D) were identified by MALDI-TOF-MS, one of which was GRP78 (point 1), another was GRP75 (point 2)

Article Snippet: Immunoblotting was carried out using rabbit anti-GRP75 antibody (Santa Cruz) or mouse anti-PreS1 antibody as indicated above and anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) conjugates.

Techniques: Binding Assay, SDS Page, Silver Staining

Journal: Brazilian Journal of Microbiology

Article Title: Identification of GRP75 as a novel PreS1 binding protein using a proteomics strategy

doi: 10.1590/S1517-838220100002000036

Figure Lengend Snippet: Protein identities and peptide masses

Article Snippet: Immunoblotting was carried out using rabbit anti-GRP75 antibody (Santa Cruz) or mouse anti-PreS1 antibody as indicated above and anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) conjugates.

Techniques: Sequencing

Journal: Brazilian Journal of Microbiology

Article Title: Identification of GRP75 as a novel PreS1 binding protein using a proteomics strategy

doi: 10.1590/S1517-838220100002000036

Figure Lengend Snippet: GRP75 binds to PreS1 in vivo. The COS7 cell lysates, co-transfected with pXJ40-PreS1 (lane 1) and pXJ40-GRP75 (lane 2) either alone or both (lane 3), were immunoprecipitated with anti-PreS1 antibody. Lysates from the transfected cell and the immunoprecipitates were subjected to Western blot analysis using the anti-GRP75 antibody

Article Snippet: Immunoblotting was carried out using rabbit anti-GRP75 antibody (Santa Cruz) or mouse anti-PreS1 antibody as indicated above and anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) conjugates.

Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot

Journal: Human molecular genetics

Article Title: Decreased chondrocyte proliferation and dysregulated apoptosis in the cartilage growth plate are key features of a murine model of epiphyseal dysplasia caused by a matn3 mutation

doi: 10.1093/hmg/ddm121

Figure Lengend Snippet: The accumulation of mutant matrilin-3 elicits an unfolded protein response in chondrocytes. (A) mRNA levels of the UPR-associated chaperones (BiP and GRP94; see methods for primer sequences) were determined using qRT-PCR analysis of mRNA isolated from mutant and wild-type chondrocytes at day 4. The graph indicates the relative increase in levels of mRNA in mutant chondrocytes when compared with wild-type chondrocytes and in both cases the level of mRNA was normalized against 18s RNA. BiP and Grp94 mRNA levels were >2- and >4-fold increased, respectively in mutant mice [n = 3 mice; three separate experiments in duplicate, independent t-test, P < 0.05 (*) or P < 0.01 (**)]. (B) Total cellular protein isolated from equal numbers of wild-type or mutant chondrocytes (2 × 105 cells) was analysed by SDS-PAGE and Western blot using antibodies raised against BiP (approximately 76 kDa) and Grp94 (approximately 94 kDa), the anti-Grp94 antibody consistently detected an non-specific band. Protein levels of both chaperones were elevated in mice homozygous for the mutation at day 4. (C) The relative levels of CHOP mRNA in mutant chondrocytes was determined by qRT-PCR. At day 3 and 5 there was no obvious upregulation of CHOP expression in mutant chondrocytes when compared with wild-type (n = 3 mice per genotype; three separate experiments, independent t-test).

Article Snippet: Antibodies to key chaperones associated with the unfolded protein response were used at 1:500 dilutions, namely BiP (Santa Cruz) and Grp94 (Santa Cruz).

Techniques: Mutagenesis, Quantitative RT-PCR, Isolation, SDS Page, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Distinct Early Molecular Responses to Mutations Causing vLINCL and JNCL Presage ATP Synthase Subunit C Accumulation in Cerebellar Cells

doi: 10.1371/journal.pone.0017118

Figure Lengend Snippet: A. Representative micrographs of wild-type, Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells immunostained with antibody recognizing the mitochondrial matrix protein, grp75, are shown. Note the elongated appearance of mitochondria in homozygous Cb Cln3 Δex7/8 and Cb Cln6 nclf cells, compared to wild-type cells in the zoomed insets. A lower magnification of a representative field of cells is also shown to demonstrate the subconfluent culture conditions. The altered mitochondrial morphology was also quantified by automated image analysis, showing a significantly reduced circularity index of the labeled mitochondria in the mutant cells, compared to wild-type cells (see ). Wild-type panel is representative of both Cb Cln3 +/+ and Cb Cln6 +/+ cell lines. Heterozygous cell lines were indistinguishable from wild-type (not shown). All images were captured at 40× magnification and were taken on the same day with identical settings. B. The bar graph depicts relative total cellular ATP levels in wild-type, heterozygous, or homozygous Cb Cln3 Δex7/8 and Cb Cln6 nclf cells, determined using the CellTiter-GLO® Luminescent Cell assay. Relative luciferase units were normalized to the wild-type cell lines and were pooled from 2–3 independent assays per cell line, each tested in 3–10 wells per assay. For reference, absolute RLUs for Cb Cln3 +/+ and Cb Cln6 +/+ cell lines were 1995830±27506 and 626172±151671, respectively. *, p≤.01 in a Student's t-test; n.s. = not significant.

Article Snippet: Commercial antibodies used were anti-nestin (Rat 401, Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences), anti-GFAP (cat#Z0334, Dako Corporation), anti-PDI (H-160, cat#sc-20132, Santa Cruz Biotechnology), anti-GM130 (cat#G65120, BD Transduction Laboratories), anti-tubulin (Sigma), anti-BiP (Abcam), anti-EEA1 (C-15, cat#sc-6414, Santa Cruz Biotechnology), anti-Rab7 (C-19, cat#sc-6563, Santa Cruz Biotechnology), anti-Lamp 1 (1D4B, cat#sc-19992, Santa Cruz Biotechnology), anti-Grp75 (cat#SPS-825, Stressgen).

Techniques: Labeling, Mutagenesis, Luciferase

Journal: PLoS ONE

Article Title: Distinct Early Molecular Responses to Mutations Causing vLINCL and JNCL Presage ATP Synthase Subunit C Accumulation in Cerebellar Cells

doi: 10.1371/journal.pone.0017118

Figure Lengend Snippet: A. Representative micrographs of confluency aged wild-type, Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and the mitochondrial marker, grp75 (red). Note the large accumulations of subunit c immunostain (white arrows) in both Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells, but which are not common in the wild-type cells. Moderate overlap of the subunit c and grp75 immunostains is observed in wild-type cells (yellow in overlay), but little to no overlap is seen in Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells. Also, again note the elongated mitochondrial morphology revealed by the grp75 immunostain in Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells, compared to wild-type cells. B. Representative micrographs of confluency aged wild-type, Cb Cln3 Δex7/8/Δex7/8 , and Cb Cln6 nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and Lamp 1 (red). Limited overlap of subunit c and Lamp 1 immunostain is observed in wild-type cells (yellow in overlay), but Lamp 1 strongly, though not perfectly, overlaps with the accumulated subunit c in Cb Cln3 Δex7/8/Δex7/8 and Cb Cln6 nclf/nclf cells. Note that in confluency aged cultures, the Lamp 1-labeled compartment appears expanded and/or aggregated in the mutant cells, which was not observed under sub-confluent culture conditions (not shown). A,B. Insets provide a zoomed view of the degree of immunostain overlap (yellow). Blue = DAPI stain. Images were captured with a 40X objective and, for like stains, were taken on the same day with identical settings.

Article Snippet: Commercial antibodies used were anti-nestin (Rat 401, Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences), anti-GFAP (cat#Z0334, Dako Corporation), anti-PDI (H-160, cat#sc-20132, Santa Cruz Biotechnology), anti-GM130 (cat#G65120, BD Transduction Laboratories), anti-tubulin (Sigma), anti-BiP (Abcam), anti-EEA1 (C-15, cat#sc-6414, Santa Cruz Biotechnology), anti-Rab7 (C-19, cat#sc-6563, Santa Cruz Biotechnology), anti-Lamp 1 (1D4B, cat#sc-19992, Santa Cruz Biotechnology), anti-Grp75 (cat#SPS-825, Stressgen).

Techniques: Marker, Labeling, Mutagenesis, Staining

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