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

Article Title: Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway

doi: 10.4049/jimmunol.1600126

Figure Lengend Snippet: (a, b, c) Expression and quantification of enhanced PGP9.5+ sensory nerves (green) co-expressing GRP (red) in the epidermis; and (d, e) Expression and quantification of GRP+ (red) neurons in Tg(+) DRG by IF. (f, k) Increased expression and numbers of GRP+ cells (red) in the dermis of Tg(+) skin; (g) GRP+ cells (red) co-expressing MBP+ eosinophils (green), (h) GRP+ cells (green) co-expressing Tryptase+ mast cells (red), and (i) GRP+ cells (red) co-expressing CD11b+ DCs/Macrophages (green) by IF. (j) GRP Receptor (GRPR) (red) inducibly expressed on Tg(+) keratinocytes, arrows, not on Tg(−) keratinocytes by IF. (l) Increased GRP+ cells in Tg(+) AD correlated with AD severity and (m) with chronic pruritus in Tg(+) mice. (n) Expression of IL-31 in the skin by qPCR, in acute AD (A) and chronic AD (C). (n=7–8 mice each group; *p<0.05, **p<0.01, and ***p<0.001. HPF: high power field).

Article Snippet: Acetone-fixed frozen sections were blocked with 10% donkey serum (Sigma-Aldrich) for 1 hr and incubated with goat anti-PGP9.5 (sc-23852; Santa Cruz Biotechnology) and rabbit anti-GRP antibody (ImmunoStar,1:1000) (the antibody reacts with human and mouse) ( 23 , 26 ) at 4°C overnight.

Techniques: Expressing

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

Article Title: Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway

doi: 10.4049/jimmunol.1600126

Figure Lengend Snippet: (a, b) IHC and quantification of dermal PGP9.5+ afferents. (c) Increased GRP+ dermal cells in AD (Normal subject skin (Nl) and lesional skin (AD) from patients), (**p<0.01, and ***p<0.001; HPF: high power field).

Article Snippet: Acetone-fixed frozen sections were blocked with 10% donkey serum (Sigma-Aldrich) for 1 hr and incubated with goat anti-PGP9.5 (sc-23852; Santa Cruz Biotechnology) and rabbit anti-GRP antibody (ImmunoStar,1:1000) (the antibody reacts with human and mouse) ( 23 , 26 ) at 4°C overnight.

Techniques:

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: Oncology Letters

Article Title: Regulation of C6 glioma cell migration by thymol

doi: 10.3892/ol.2016.4237

Figure Lengend Snippet: Western blot analysis of PKCα and ERK1/2 phosphorylation. (A) C6 glioma cells were cultured in serum-free medium for 24 h and then in the presence of thymol (3–30 µM) for 1 h, followed by exposure to 10% FBS for 24 h. Cell lysates were separated by 12% polyacrylamide gel electrophoresis and the specific protein bands were labeled using antibodies. The band intensities were measured and reflect the effect of thymol on the phosphorylation of (B) PKCα and (C) ERK1/2. These values were normalized against total PKCα (T-PKCα), total ERK1/2 (T-ERK1/2) and GAPDH. Data are expressed as the mean ± standard error (n=3). Cell response to FBS is expressed as 100%. *P<0.05 vs. FBS-stimulated cells in the absence of thymol. PKCα, protein kinase Cα; ERK1/2, extracellular signal-regulated kinases 1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FBS, fetal bovine serum.

Article Snippet: Other antibodies, such as rabbit polyclonal anti-rat anti-ERK1/2 (catalog no., sc-94), mouse monoclonal anti-rat anti-P-ERK1/2 (catalog no., sc-7383), rabbit polyclonal anti-rat anti-MMP2 (catalog no., sc-10736) and goat polyclonal anti-rat anti-MMP9 (catalog no., sc-6840), were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Techniques: Western Blot, Cell Culture, Polyacrylamide Gel Electrophoresis, Labeling

Journal:

Article Title: Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity

doi:

Figure Lengend Snippet: Characterization of heterozygous ORP150-deficient mice and transgenic mice. (a and f) ELISA analysis of ORP150 was performed in protein extracts prepared from systemic organs (a) or hippocampus 12 hours after the administration of kainate (f) (0–0.4 μg) of either heterozygous ORP150-deficient mice (ORP150–/+), wild-type littermates (ORP150+/+), or Tg PD-ORP150 mice (Transgenic). The mean ± SD is shown (n = 6). (b–e) Baseline expression of ORP150 was assessed by immunohistochemical analysis using anti-ORP150 Ab; (b–d) ×80, and (e) ×400. (g) Expression of Glu-R6 assessed immunohistochemically in each type of mice (×200). Expression of GRP78 (h) and GRP94 (i) was assessed by immunohistochemical analysis using specific Ab’s 12 hours after the intrahippocampal administration of kainate (0.1 μg; ×40).

Article Snippet: Sections were processed for cresyl violet staining or immunohistochemistry using either rabbit anti-human ORP150 (5 μg/ml) ( 3 – 5 ), monoclonal mouse anti-MAP2 (Sigma Chemical Co., St. Louis, Missouri, USA; 1:1,000 dilution), goat anti-GFAP (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), goat anti–Glu-R6 (1:100 dilution; Santa Cruz Biotechnology Inc.), rabbit anti–GRP78 (1:500 dilution; StressGen Biotecnologies Corp., Victoria, British Columbia, Canada), or rabbit anti–GRP94 (1:500 dilution; StressGen Biotecnologies Corp.).

Techniques: Transgenic Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemical 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