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: Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

doi: 10.1093/hmg/ddaa243

Figure Lengend Snippet: GDAP1 interacts with SYNTAXIN 17 (STX17) and LC3 in MAMs. ( A ) Negative interactions by co-IP assays of GDAP1 with DRP1 (mitochondrial fission in MAMs), ACSL1 (fatty acid metabolism in MAMs), GRP75 (Ca 2+ channel in MAMs), BECLIN-1 (ER-mitochondria tethering and autophagosome formation), ATG4 (unique redox sensor essential for maturation of autophagosomes) and RAB7 (trafficking, maturation, and fusion of endocytic and autophagic vesicles). ( B and C ) Co-IP assay of endogenous GDAP1 and STX17 (B) or LC3 (C) in SH-SY5Y cells. ( D and E ) Representative images of the interaction between GDAP1 and STX17 (D) or LC3 (E) in SH-SY5Y cells by PLA from 3 independent experiments. Scale bar: 10 μm. ( F ) Western blot of subcellular fractions from SH-SY5Y and G4 cells and quantification of relative protein levels in MAMs fraction (three or four independent experiments). C, cytosol; ER, endoplasmic reticulum; pM, pure mitochondria; MAM, mitochondria associated membranes. Data information: In (F), data represent mean ± SD and individual values are displayed as dots. ANOVA followed by Sidak’s post hoc test.

Article Snippet: The following antibodies were used: ACSL1 rabbit antibody (Cell Signaling, #4047), ATG4B mouse antibody (MBL, M134-3), ATG4B rabbit antibody (Abcam, ab154843), BECLIN-1 rabbit antibody (Cell Signaling #3738), β-ACTIN mouse antibody (Sigma-Aldrich, A5316), TUBB3 rabbit antibody (Sigma-Aldrich, T2200), TUBB3 mouse antibody (Promega, G7121), DRP1 mouse antibody (BD Biosciences, 611113), FACL-4 mouse antibody (Santa Cruz, sc-365230), GDAP1 rabbit antibody (Sigma-Aldrich, HPA024334), GDAP1 mouse antibody (Abcam, ab194493), GRP75 rabbit antibody (Santa Cruz, sc-13967), HA-probe mouse antibody (Santa Cruz, sc-7392), LAMP-1 rabbit antibody (Sigma-Aldrich, L1418 and Abcam ab24170), LAMP-1 mouse antibody (DSHB, H4A3), LC3 mouse antibody (LifeSpan Biosciences, LS-C165694), LC3B rabbit antibody (Sigma-Aldrich, L7543), MITOFUSIN-2 rabbit antibody (Sigma-Aldrich, M6319), PIK fyve mouse antibody (Santa Cruz, sc-100408), p62/SQSTM1 rabbit antibody (Sigma-Aldrich, P0067), RAB7 mouse antibody (Sigma-Aldrich, R8779), SYNTAXIN 17 rabbit antibody (Sigma-Aldrich, HPA001204), TFEB rabbit antibody (LifeSpan Biosciences, LS-C353036), TOM20 mouse antibody (BD Biosciences, 612278), V5 rabbit antibody (Sigma-Aldrich,V8137) and Alexa fluorophore-conjugated secondary antibodies from Molecular Probes (Invitrogen).

Techniques: Co-Immunoprecipitation Assay, Western Blot

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