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  • 99
    Thermo Fisher grp78 bip
    Effect of EEAC on endoplasmic-reticulum (ER). T47D cells were treated with EEAC (25 and 50 μg/mL) for 48 h. ( A ) Cells were collected and stained with Fluo-3 AM for the determination of Ca 2+ concentration and analyzed using flow cytometry. ( B ) Expression of ER stress sensor proteins IRE1 (inositol-requiring enzyme 1α), PERK (pancreatic endoplasmic reticulum kinase), and ATF-6 (activating transcription factor 6), as well as ER stress marker <t>GRP78/Bip</t> and CCAAT-enhancer-binding protein homologous protein (CHOP), was determined by Western blot assay. Actin was used as the loading control. ( C ) Confocal microscopy image of GRP78/Bip (red) and CHOP (green) stained T47D cells were treated with EEAC (50 μg/mL) for 48 h. Cells were counterstained with DAPI to label cells nuclei (blue). All the results are presented as mean ± SD of at least three experiments, ** p
    Grp78 Bip, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti grp78 bip et 21
    Effect of EEAC on endoplasmic-reticulum (ER). T47D cells were treated with EEAC (25 and 50 μg/mL) for 48 h. ( A ) Cells were collected and stained with Fluo-3 AM for the determination of Ca 2+ concentration and analyzed using flow cytometry. ( B ) Expression of ER stress sensor proteins IRE1 (inositol-requiring enzyme 1α), PERK (pancreatic endoplasmic reticulum kinase), and ATF-6 (activating transcription factor 6), as well as ER stress marker <t>GRP78/Bip</t> and CCAAT-enhancer-binding protein homologous protein (CHOP), was determined by Western blot assay. Actin was used as the loading control. ( C ) Confocal microscopy image of GRP78/Bip (red) and CHOP (green) stained T47D cells were treated with EEAC (50 μg/mL) for 48 h. Cells were counterstained with DAPI to label cells nuclei (blue). All the results are presented as mean ± SD of at least three experiments, ** p
    Anti Grp78 Bip Et 21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hspa5  (Abcam)
    93
    Abcam hspa5
    Western blot. Cross-validation of 2DE gel data by Western blot of HSP90B1, HSP60, <t>HSPA5</t> and HSPA8 in control (CON) and TGF-β2 treated IECs (TGF). (A) Representative blots. (B) Fold-changes of HSPs in TGF, compared with CON analyzed by densitometry and Student’s t-test. Values were means ± SEM (n = 5 in each treatment). * P
    Hspa5, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc hspa5 grp78 bip
    Positions of <t>Grp78/BiP</t> positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm
    Hspa5 Grp78 Bip, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 87/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stressgen Biotechnologies hspa5 bip
    Positions of <t>Grp78/BiP</t> positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm
    Hspa5 Bip, supplied by Stressgen Biotechnologies, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals grp78 hspa5 antibody
    Positions of <t>Grp78/BiP</t> positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm
    Grp78 Hspa5 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti hspa5 grp78 bip
    Positions of <t>Grp78/BiP</t> positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm
    Anti Hspa5 Grp78 Bip, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem grp78 hspa5 bip
    Positions of <t>Grp78/BiP</t> positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm
    Grp78 Hspa5 Bip, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals anti grp78
    Effects of HFD on markers of adipocyte function and injury in WT and NOX2KO mice. Expression of ( A ) PPARγ, ( B ) adiponectin, ( C ) GADD153/CHOP, and ( D ) <t>GRP78</t> were evaluated in tissue homogenates prepared from epididymal adipose depots. Data were
    Anti Grp78, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology bip grp78
    Microscopic analysis of the distribution of <t>BiP/GRP78</t> and CHOP/Gadd153 in immunohistochemically stained cochleae (SP, ×400). (a-1–a-3) Cells that were positively stained for BiP/GRP were located in the organ of Corti, the lateral wall, and the spiral ganglion, respectively, in the 1d group (positive expression: immuoreactivity is brown versus the blue hematoxylin stain, indicated by ↑). (b-1–b-3) Cells that were positively stained for CHOP/Gadd153 were located in the same regions as those shown in (a), in the 1d group (positive expression: immunoreactivity is tan or medium-brown particles versus hematoxylin-stain, indicated by ↑). (c-1–c-3) Negative control incubated with PBS instead of the primary antibodies (organ of Corti, the lateral wall, and the spiral ganglion, respectively)
    Bip Grp78, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson grp78 bip
    <t>Grp78/BiP</t> protein identification and quantitation using label-free spectral counting-based mass spectrometry. (A) Workflow of label-free spectral counting-based protein quantitative analysis using LC-MS/MS. Protein samples were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and gel pieces excised at ~80 kDa for the purpose of in-gel trypsin digestion and LC-MS/MS analysis. Protein quantitative data analysis was conducted using spectral counting and interpreted by normalized total spectra numbers. (B) Grp78/BiP protein identification and peptide coverage using LC-MS/MS. (C) Representative mass-to-charge ratio spectrum and b-/y ions fragmentation of Grp78/BiP peptide (highlighted in B ). (D) Protein quantitative data analysis using the ratio of normalized total spectra numbers of Grp78/BiP to a house keeping protein GAPDH. Three independent muscles from 120–140d old wild type (WT) and ALS-Tg mice were analyzed. * p
    Grp78 Bip, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson bip grp78
    Deletion of Tlr2 inhibits increased cytokine sensitivity in Trim58 -deficient myeloid cells. ( A ) Assessment of TLR2, mature IL-1β, arginase-1, and <t>BIP/GRP78</t> protein synthesis in peritoneal myeloid (PM) cells from male Trim58 +/+ and Trim58 −/− mice ( n = 4–6 per genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by immunoblot (IB) analysis. ( B ) Assessment of mature IL-1β protein synthesis in PM cells from male Tlr2 −/− , Trim58 −/− and Trim58 −/− Tlr2 −/− mice ( n = 4–6/genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by IB analysis. Blots were reprobed with anti-GAPDH to confirm equal loading. Right margin, molecular size markers (kDa). Representative results of at least two independent experiments are shown.
    Bip Grp78, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human grp78 hspa5 antibody
    Deletion of Tlr2 inhibits increased cytokine sensitivity in Trim58 -deficient myeloid cells. ( A ) Assessment of TLR2, mature IL-1β, arginase-1, and <t>BIP/GRP78</t> protein synthesis in peritoneal myeloid (PM) cells from male Trim58 +/+ and Trim58 −/− mice ( n = 4–6 per genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by immunoblot (IB) analysis. ( B ) Assessment of mature IL-1β protein synthesis in PM cells from male Tlr2 −/− , Trim58 −/− and Trim58 −/− Tlr2 −/− mice ( n = 4–6/genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by IB analysis. Blots were reprobed with anti-GAPDH to confirm equal loading. Right margin, molecular size markers (kDa). Representative results of at least two independent experiments are shown.
    Human Grp78 Hspa5 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore bip grp78
    MCD medium-induced ER stress activation in McA cells. Total cell lysate (70 μg of protein) was analyzed by immunoblotting for phospho-JNK, phospho-PERK, phospho-eIF2α, CHOP, <t>BiP/GRP78,</t> and GAPDH. A , McA cells were treated as described
    Bip Grp78, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology grp78 bip
    ERp72 and <t>GRP78/BiP</t> protein are elevated and ERp72 oxidized state is increased in AdDNPerk 832/13 beta cells . A. Triplicate samples were probed with antibodies to ERp72, ERp57, BiP and actin, which served as the loading control. B. ERp72 expression was increased in islets from P2 Perk KO mice. Samples are from duplicate mice for each genotype. C. At 24 hr or 36 hr post-transduction, protein samples were isolated and treated with AMS to differentiate the reduced and oxidized forms of ERp72 and ERp57 on PAGE gels. Western blots showed increased oxidized isoforms of both ERp72 and ERp57 in 832/13 cells infected with AdDNPerk compared to cells infected with AdLacZ .
    Grp78 Bip, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc anti hspa5 bip
    ERp72 and <t>GRP78/BiP</t> protein are elevated and ERp72 oxidized state is increased in AdDNPerk 832/13 beta cells . A. Triplicate samples were probed with antibodies to ERp72, ERp57, BiP and actin, which served as the loading control. B. ERp72 expression was increased in islets from P2 Perk KO mice. Samples are from duplicate mice for each genotype. C. At 24 hr or 36 hr post-transduction, protein samples were isolated and treated with AMS to differentiate the reduced and oxidized forms of ERp72 and ERp57 on PAGE gels. Western blots showed increased oxidized isoforms of both ERp72 and ERp57 in 832/13 cells infected with AdDNPerk compared to cells infected with AdLacZ .
    Anti Hspa5 Bip, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti grp78 bip
    Retinal ER stress and UPR evaluation in Wfs1 −/− mice. Immunoblots (A) detected <t>GRP78/BiP,</t> PDI, IRE1, and beta actin in protein lysates of 12 month old Wfs1 +/+ (n = 3) and Wfs1 −/− (n = 3) mouse retinas and in mouse NIH3T3 fibroblasts treated with thapsigargin. Mean relative quantities for each protein according to Wfs1 genotype were obtained after normalization with beta actin values. Significance (*) is indicated when p
    Anti Grp78 Bip, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam recombinant bip protein
    Retinal ER stress and UPR evaluation in Wfs1 −/− mice. Immunoblots (A) detected <t>GRP78/BiP,</t> PDI, IRE1, and beta actin in protein lysates of 12 month old Wfs1 +/+ (n = 3) and Wfs1 −/− (n = 3) mouse retinas and in mouse NIH3T3 fibroblasts treated with thapsigargin. Mean relative quantities for each protein according to Wfs1 genotype were obtained after normalization with beta actin values. Significance (*) is indicated when p
    Recombinant Bip Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals rabbit anti grp78
    Sialic acids and <t>GRP78</t> act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p
    Rabbit Anti Grp78, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech anti hspa5 grp78
    Sialic acids and <t>GRP78</t> act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p
    Anti Hspa5 Grp78, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    StressMarq anti hspa5 grp78
    Sialic acids and <t>GRP78</t> act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p
    Anti Hspa5 Grp78, supplied by StressMarq, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti bip
    SMER28 induced cocompartmentalization of <t>APP-CTF</t> and LC3-II. A ) N2a-APP cells were treated for 6 h with SMER28 (50 μM), and whole-cell lysates were fractionated by sucrose gradient. An aliquot of each of the 12 fractions recovered was analyzed by SDS-PAGE and Western blotting for APP-CTF, LC3, γ-adaptin, and <t>Bip.</t> B ) N2a-APP cells were transfected with an LC3-EGFP-containing plasmid; at 16 h post-transfection, cells were treated for 6 h with SMER28 (50 μM). Cells were fixed and immunostained with RU-369, an APP C-terminal antibody, or APLP1 antibody and imaged by confocal microscopy.
    Anti Bip, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bip grp78
    SMER28 induced cocompartmentalization of <t>APP-CTF</t> and LC3-II. A ) N2a-APP cells were treated for 6 h with SMER28 (50 μM), and whole-cell lysates were fractionated by sucrose gradient. An aliquot of each of the 12 fractions recovered was analyzed by SDS-PAGE and Western blotting for APP-CTF, LC3, γ-adaptin, and <t>Bip.</t> B ) N2a-APP cells were transfected with an LC3-EGFP-containing plasmid; at 16 h post-transfection, cells were treated for 6 h with SMER28 (50 μM). Cells were fixed and immunostained with RU-369, an APP C-terminal antibody, or APLP1 antibody and imaged by confocal microscopy.
    Bip Grp78, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti grp78
    Melatonin suppressed KA-induced ER stress in vivo. (A) Expression of <t>GRP78,</t> CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (B ) Relative analysis of the expression levels of GRP78, CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (C) Relative activity of calpain in KA and/or melatonin-treated animals hippocampus. ∗∗ P
    Anti Grp78, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam grp78 protein
    Knocking-down the expression of <t>GRP78</t> does not affect the replication of JE viral RNA . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (scramble siRNA) for 48 hours. The expression level of GRP78 was measured by Western blot analysis using polyclonal antibody specific to GRP78. Transfected cells were then infected with JEV at an MOI of 1. At 24 hour post-infection, the cell lysates were collected to measure the JEV replication using antibodies specific to NS1 and/or NS5. Two independent replicates are shown for the scramble and GRP78 siRNA conditions
    Grp78 Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech rabbit grp78 bip polyclonal
    Knocking-down the expression of <t>GRP78</t> does not affect the replication of JE viral RNA . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (scramble siRNA) for 48 hours. The expression level of GRP78 was measured by Western blot analysis using polyclonal antibody specific to GRP78. Transfected cells were then infected with JEV at an MOI of 1. At 24 hour post-infection, the cell lysates were collected to measure the JEV replication using antibodies specific to NS1 and/or NS5. Two independent replicates are shown for the scramble and GRP78 siRNA conditions
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    Abcam grp78 polyclonal antibody
    Sialic acids and <t>GRP78</t> act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 <t>polyclonal</t> antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p
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    Proteintech grp78
    Alteration of the expressions of apoptosis-associated mitochondrial and ER stress proteins in the bladder among different experimental groups. ( A – E ) The apoptosis of bladder cells as detected by TUNEL, (FITC, green) and DAPI staining (blue) for UL and SL. There were increases in TUNEL - positive nuclei (white arrows) in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. Scale bar = 100 μm. ( F – I ) The expression levels of pro-apoptotic and anti-apoptotic proteins in the bladder tissue by western blots. ( F , G ) Quantifications of the ER stress protein expressions to β-actin. The expression levels of <t>GRP78,</t> CHOP and caspase-12 slightly were increased in the MetS group, and significantly promoted in the MetS + OVX and MetS + OVX + EGCG groups. ( H , I ) Quantifications of mitochondrial protein expressions to β-actin. The expression of Bcl-2 was decreased in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. In contrast, the pro-apoptosis expressions were meaningfully increased in those groups. Results were normalized as the control = 100%. Values represented the mean ± SD for n = 8. * P
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    Abcam rabbit anti grp78
    The combination of PTN and three required binding partners promotes DIPG invasion toward SVZ hNPC CM . (A) No single candidate recombinant protein significantly increased DIPG invasion compared to unconditioned hNPC media. (B) The combination of four factors: PTN, SPARC, SPARCL1, and HSP90B, was sufficient for the full invasion-promoting effect toward SVZ hNPC CM. No combination of two or three factors was sufficient. All experiments performed with n = 3 replicates/wells in SU-DIPG-XIII FL cells and analyzed by one-way ANOVA with Tukey post hoc adjustment for multiple comparisons (A) or Dunnett post hoc adjustment for multiple comparisons to either SVZ hNPC CM or the combination of all 4 factors (B). (C) All four proteins coeluted at approximately the 212 kDa size expected for a complex of all four proteins by size exclusion chromatography. (D) All four proteins copurified together in immunoprecipitation reactions for any one of the four proteins, more so than with a control IgG. Three control proteins also present in SVZ hNPC CM: <t>GRP78,</t> IGFBP2, and BCAN, did not copurify with any of the four proteins. ) invade preferentially toward the combination of four proteins similarly to toward human SVZ NPC CM, compared to unconditioned hNPC media. All experiments performed with n = 3 replicates/wells and analyzed by unpaired, two-tailed Student’s t-tests for comparison between unconditioned and conditioned hNPC media. Data shown as mean ± SEM. *p
    Rabbit Anti Grp78, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti bip grp78
    Expression patterns of unfolded protein response ( UPR ) signal genes in cumulus‐oocyte complexes ( COC s), denuded oocyte ( DO ), and cumulus cells ( CC ) on porcine oocytes in vitro maturation ( IVM ) (22 and 44 h). A, The mRNA levels of activated UPR signal transcription factors ( <t>Bip/Grp78</t> and Atf4 ) on maturing COC s, DO , and CC of porcine IVM process (metaphase, M I; 22 h and metaphase, M II ; 44 h) were measured by reverse transcription‐polymerase chain reaction ( PCR ) ( RT ‐ PCR ) analysis. Relative folds of Bip/Grp78 and Atf4 were obtained by normalizing the signals for Gapdh . B‐C, Western blotting results of Bip/Grp78, ATF 4, P50 ATF 6, and CHOP in DO , COC s, and CC were compared at the M I (22 h) and M II (44 h) stages of pig oocyte maturation. Relative folds of UPR marker protein levels were obtained by normalizing the signals for β‐Actin. D, Immunohistochemistry ( IHC ) staining of P50 ATF 6 in pig ovaries with different follicle sizes (1‐2 mm, small; 3‐4 mm, middle; and 5‐6 mm, large). IHC staining for P50 ATF 6 is detected using specific P50 ATF 6 antibody in in vivo ‐ maturing oocytes of pig ovary follicles. O = immature oocyte, Scale bar = 200 μm. Histograms represent values of densitometry analysis obtained using ImageJ software. Data in the bar graph are means ± SEM / SD of three independent experiments (per 50 DO s and 30 COC s). *** P
    Anti Bip Grp78, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti grp78 bip
    Cab45S increases the <t>GRP78/BiP</t> protein level. ( a and d ) Western blots of GRP78/BiP and two other ER molecular chaperones, PDI and calnexin, in stable Cab45S-knockdown ( a ) or Cab45S-overexpressed ( d ) PANC-1 cell lines. Numbers represent different cell lines. ( b and c ) Western blots ( b ) and quantification ( c ) of GRP78/BiP in Cab45S-knockdown and control (shNC, scrambled shRNA) HeLa cells treated with TM (2 μ g/ml) for the indicated periods. GAPDH was used as a loading control. ( e ) Quantitative real-time PCR of the relative GRP78/BiP mRNA expression levels in Cab45S-knockdown and control HeLa cells treated with TM for the indicated times ( n =3). ( f ) Western blots of GRP78/BiP in Cab45S-knockdown and control HeLa cells treated with TM (2 μ g/ml, 4 h) followed by cycloheximide (Chx; 100 μ M) for the indicated periods. For c and e , data are presented as mean±S.E.M. ** P
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    Image Search Results


    Effect of EEAC on endoplasmic-reticulum (ER). T47D cells were treated with EEAC (25 and 50 μg/mL) for 48 h. ( A ) Cells were collected and stained with Fluo-3 AM for the determination of Ca 2+ concentration and analyzed using flow cytometry. ( B ) Expression of ER stress sensor proteins IRE1 (inositol-requiring enzyme 1α), PERK (pancreatic endoplasmic reticulum kinase), and ATF-6 (activating transcription factor 6), as well as ER stress marker GRP78/Bip and CCAAT-enhancer-binding protein homologous protein (CHOP), was determined by Western blot assay. Actin was used as the loading control. ( C ) Confocal microscopy image of GRP78/Bip (red) and CHOP (green) stained T47D cells were treated with EEAC (50 μg/mL) for 48 h. Cells were counterstained with DAPI to label cells nuclei (blue). All the results are presented as mean ± SD of at least three experiments, ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Antrodia cinnamomea, a Treasured Medicinal Mushroom, Induces Growth Arrest in Breast Cancer Cells, T47D Cells: New Mechanisms Emerge

    doi: 10.3390/ijms20040833

    Figure Lengend Snippet: Effect of EEAC on endoplasmic-reticulum (ER). T47D cells were treated with EEAC (25 and 50 μg/mL) for 48 h. ( A ) Cells were collected and stained with Fluo-3 AM for the determination of Ca 2+ concentration and analyzed using flow cytometry. ( B ) Expression of ER stress sensor proteins IRE1 (inositol-requiring enzyme 1α), PERK (pancreatic endoplasmic reticulum kinase), and ATF-6 (activating transcription factor 6), as well as ER stress marker GRP78/Bip and CCAAT-enhancer-binding protein homologous protein (CHOP), was determined by Western blot assay. Actin was used as the loading control. ( C ) Confocal microscopy image of GRP78/Bip (red) and CHOP (green) stained T47D cells were treated with EEAC (50 μg/mL) for 48 h. Cells were counterstained with DAPI to label cells nuclei (blue). All the results are presented as mean ± SD of at least three experiments, ** p

    Article Snippet: Cells were incubated with the acetyl-histone H3, H4, GRP78/bip, and CHOP for 2 h and secondary antibodies for 1 h in a ratio of 1:1000 (Alexa Fluor 586-conjugated goat anti-mouse/rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse/rabbit IgG, Life Technologies, Carlsbad, CA, USA) at normal temperature.

    Techniques: Staining, Concentration Assay, Flow Cytometry, Cytometry, Expressing, Marker, Binding Assay, Western Blot, Confocal Microscopy

    Western blot. Cross-validation of 2DE gel data by Western blot of HSP90B1, HSP60, HSPA5 and HSPA8 in control (CON) and TGF-β2 treated IECs (TGF). (A) Representative blots. (B) Fold-changes of HSPs in TGF, compared with CON analyzed by densitometry and Student’s t-test. Values were means ± SEM (n = 5 in each treatment). * P

    Journal: PLoS ONE

    Article Title: Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

    doi: 10.1371/journal.pone.0117608

    Figure Lengend Snippet: Western blot. Cross-validation of 2DE gel data by Western blot of HSP90B1, HSP60, HSPA5 and HSPA8 in control (CON) and TGF-β2 treated IECs (TGF). (A) Representative blots. (B) Fold-changes of HSPs in TGF, compared with CON analyzed by densitometry and Student’s t-test. Values were means ± SEM (n = 5 in each treatment). * P

    Article Snippet: Anti-HSP60, HSPA5, HSPA8, HSP90B1 antibodies (Abcam, Cambridge, UK) were used.

    Techniques: Western Blot, Two-Dimensional Gel Electrophoresis

    HKH40A affects transcription of GRP78/BiP. ( a ) qPCR analysis of BiP transcripts in HCT-116 and HT-29 cells untreated or treated with 100 nM HKH40A for 24 h. ( b ) Promoter activity in HCT-116 and HT-29 cells transfected with pGL-BiP-P plasmid and treated with 100 nM HKH40A for 24 h (fold change relative to pGL-4.10 empty vector transfected cells)

    Journal: Cell Death & Disease

    Article Title: HKH40A downregulates GRP78/BiP expression in cancer cells

    doi: 10.1038/cddis.2014.203

    Figure Lengend Snippet: HKH40A affects transcription of GRP78/BiP. ( a ) qPCR analysis of BiP transcripts in HCT-116 and HT-29 cells untreated or treated with 100 nM HKH40A for 24 h. ( b ) Promoter activity in HCT-116 and HT-29 cells transfected with pGL-BiP-P plasmid and treated with 100 nM HKH40A for 24 h (fold change relative to pGL-4.10 empty vector transfected cells)

    Article Snippet: After 1 h blocking of nonspecific binding sites using 5% bovine serum albumin (BSA), cells were stained 1 h with the primary antibody to GRP78/BiP (Rabbit polyclonal, ab21685, Abcam) diluted 1 : 500 in 5% BSA then washed with PBS-T, stained with secondary antibody Alexa 594 (1 : 1000 in 5% BSA) for 1 h followed by final PBS-T washes and examined using a Zeiss LC510 confocal microscope.

    Techniques: Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Plasmid Preparation

    HKH40A directly binds BiP and affects its stability. ( a ) Colocalization of HKH40A with BiP. HCT-116 cells were treated with 100 nM HKH40A for 3 h, fixed and immunostained for GRP78. The drug with intrinistic ability to fluoresce binds to DNA and emits very bright fluorescence. Fluorescence is also visible in cytoplasm. Immunostaining of cells with GRP78 shows perinuclear and cytoplasmic localization. Some overlap of the two signals in the perinuclear region can be noted in the orthogonal views of the Z-stacks, scale bar 10 μ m. ( b ) Direct interaction of HKH40A with recombinant human GRP78/BiP. Microscale thermophoresis analysis revealed direct interaction of HKH40A with recombinant human GRP78/BiP. Analysis was performed using the intrinsic ability of HKH40A to fluoresce. Purified recombinant human K-Ras (1–166) was used as a negative control (in red). Its addition caused no change in the thermophoretic mobility of the compound. ( c ) Proteosomal degradation is involved in HKH40A downregulation of BiP. HCT-116 and HT-29 cells were treated with 100 nM HKH40A, 10 μ M MG132 or a combination of both agents for 6 h, then drugs were removed, cells were cultured in a drug-free medium for up to 24 h, collected and analyzed by western blot. ( d ) Topoisomerase1 and topoisomerase2 inhibition is not responsible for the reduction of GRP78/BiP level by HKH40A. HT-29 cells were cultured for 24 h with vehicle (1), 100 nM HKH40A (2), 5 μ M CPT-11 (3), 10 μ M Etoposide (4), 250 nM adriamycin (5) and 1 μ M WMC26 (6). Total cell lysates were subjected to western blots with anti-GRP78/BiP. β -Actin was used as a loading control

    Journal: Cell Death & Disease

    Article Title: HKH40A downregulates GRP78/BiP expression in cancer cells

    doi: 10.1038/cddis.2014.203

    Figure Lengend Snippet: HKH40A directly binds BiP and affects its stability. ( a ) Colocalization of HKH40A with BiP. HCT-116 cells were treated with 100 nM HKH40A for 3 h, fixed and immunostained for GRP78. The drug with intrinistic ability to fluoresce binds to DNA and emits very bright fluorescence. Fluorescence is also visible in cytoplasm. Immunostaining of cells with GRP78 shows perinuclear and cytoplasmic localization. Some overlap of the two signals in the perinuclear region can be noted in the orthogonal views of the Z-stacks, scale bar 10 μ m. ( b ) Direct interaction of HKH40A with recombinant human GRP78/BiP. Microscale thermophoresis analysis revealed direct interaction of HKH40A with recombinant human GRP78/BiP. Analysis was performed using the intrinsic ability of HKH40A to fluoresce. Purified recombinant human K-Ras (1–166) was used as a negative control (in red). Its addition caused no change in the thermophoretic mobility of the compound. ( c ) Proteosomal degradation is involved in HKH40A downregulation of BiP. HCT-116 and HT-29 cells were treated with 100 nM HKH40A, 10 μ M MG132 or a combination of both agents for 6 h, then drugs were removed, cells were cultured in a drug-free medium for up to 24 h, collected and analyzed by western blot. ( d ) Topoisomerase1 and topoisomerase2 inhibition is not responsible for the reduction of GRP78/BiP level by HKH40A. HT-29 cells were cultured for 24 h with vehicle (1), 100 nM HKH40A (2), 5 μ M CPT-11 (3), 10 μ M Etoposide (4), 250 nM adriamycin (5) and 1 μ M WMC26 (6). Total cell lysates were subjected to western blots with anti-GRP78/BiP. β -Actin was used as a loading control

    Article Snippet: After 1 h blocking of nonspecific binding sites using 5% bovine serum albumin (BSA), cells were stained 1 h with the primary antibody to GRP78/BiP (Rabbit polyclonal, ab21685, Abcam) diluted 1 : 500 in 5% BSA then washed with PBS-T, stained with secondary antibody Alexa 594 (1 : 1000 in 5% BSA) for 1 h followed by final PBS-T washes and examined using a Zeiss LC510 confocal microscope.

    Techniques: Fluorescence, Immunostaining, Recombinant, Microscale Thermophoresis, Purification, Negative Control, Cell Culture, Western Blot, Inhibition, Cycling Probe Technology

    PARK7 interacts with R-HSPA5, the Nt-arginylated form of HSPA5. (a) Cells were engineered to stably express either p3x-FLAG or FLAG-tagged PARK7 (FLAG-PARK7) and treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated using anti-FLAG antibody, and the precipitated proteins were visualized using silver staining. (b) Cells were transiently transfected with a plasmid expressing either FLAG-PARK7 or HA-tagged HSPA5. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody (top). The presence of FLAG-PARK7 and HA-HSPA5 in the lysates was verified by immunoblotting (bottom). (c) Cells were treated with 5 ng/ml TNFSF10 for 3 h, and cell lysates were immunoprecipitated with anti-PARK7 antibody or mock antibody (rabbit IgG) followed by immunoblotting with anti-HSPA5 or anti-PARK7 antibody (top). The presence of HSPA5 and PARK7 in the lysates was verified using immunoblotting (bottom). (d) A schematic diagram in which TNFSF10 induces the Nt-arginylation of HSPA5. In this mechanism, newly synthesized HSPA5 translocates into the ER lumen, during which its signal peptide is cleaved off by the signal peptide peptidase, resulting in mature HSPA5. Our results suggest that TNFSF10 induces the cytosolic retrotranslocation and Nt-arginylation of lumenal HSPA5, resulting in cytosolic accumulation of R-HSPA5. (e) HCT116 cells were treated with 10 ng/ml TNFSF10, followed by immunoblotting of R-HSPA5, HSPA5, and ATE1. (f) HCT116 cells were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were fractionated to enrich the cytosol, mitochondria, and ER. Fractionated proteins were immunoblotted for R-HSPA5, PARK7, HSPA5, the mitochondrial channel VDAC (voltage dependent anion channel), the ER chaperone CANX (calnexin).

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: PARK7 interacts with R-HSPA5, the Nt-arginylated form of HSPA5. (a) Cells were engineered to stably express either p3x-FLAG or FLAG-tagged PARK7 (FLAG-PARK7) and treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated using anti-FLAG antibody, and the precipitated proteins were visualized using silver staining. (b) Cells were transiently transfected with a plasmid expressing either FLAG-PARK7 or HA-tagged HSPA5. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody (top). The presence of FLAG-PARK7 and HA-HSPA5 in the lysates was verified by immunoblotting (bottom). (c) Cells were treated with 5 ng/ml TNFSF10 for 3 h, and cell lysates were immunoprecipitated with anti-PARK7 antibody or mock antibody (rabbit IgG) followed by immunoblotting with anti-HSPA5 or anti-PARK7 antibody (top). The presence of HSPA5 and PARK7 in the lysates was verified using immunoblotting (bottom). (d) A schematic diagram in which TNFSF10 induces the Nt-arginylation of HSPA5. In this mechanism, newly synthesized HSPA5 translocates into the ER lumen, during which its signal peptide is cleaved off by the signal peptide peptidase, resulting in mature HSPA5. Our results suggest that TNFSF10 induces the cytosolic retrotranslocation and Nt-arginylation of lumenal HSPA5, resulting in cytosolic accumulation of R-HSPA5. (e) HCT116 cells were treated with 10 ng/ml TNFSF10, followed by immunoblotting of R-HSPA5, HSPA5, and ATE1. (f) HCT116 cells were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were fractionated to enrich the cytosol, mitochondria, and ER. Fractionated proteins were immunoblotted for R-HSPA5, PARK7, HSPA5, the mitochondrial channel VDAC (voltage dependent anion channel), the ER chaperone CANX (calnexin).

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Stable Transfection, Immunoprecipitation, Silver Staining, Transfection, Plasmid Preparation, Expressing, Synthesized

    The autophagic targeting of R-HSPA5 and SQSTM1 is impaired in PARK7-deficient cells. (a) HCT116 cells stably expressing scrambled shRNA or sh PARK7 were treated with 200 nM bafilomycin A 1 (Baf) for 6 h and immunostained for LC3-II (red) or PARK7 (green). LC3-II-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm. (b) Quantification of the number of LC3-II puncta in A. Error bars represent the mean ± SEM from each cell (*** p

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: The autophagic targeting of R-HSPA5 and SQSTM1 is impaired in PARK7-deficient cells. (a) HCT116 cells stably expressing scrambled shRNA or sh PARK7 were treated with 200 nM bafilomycin A 1 (Baf) for 6 h and immunostained for LC3-II (red) or PARK7 (green). LC3-II-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm. (b) Quantification of the number of LC3-II puncta in A. Error bars represent the mean ± SEM from each cell (*** p

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Stable Transfection, Expressing, shRNA, Confocal Microscopy

    Hypothetical model for the role of PARK7 in autophagic protein quality control. In this model, TNFSF10 causes mitochondrial misregulation and oxidative stress associated with the excessive generation of ROS. This causes the formation of cytosolic misfolded proteins that are tagged with Ub but cannot be degraded by the UPS (step 1). In response to the proteotoxicity, cells induce autophagic protein quality control, which involves the Nt-arginylation of the ER-resident HSPA5 (step 2). In parallel, PARK7 is oxidized (step 3). The resulting PARK7 binds R-HSPA5 as its cofactor/co-chaperone that facilitates the ability of association with binding Ub-tagged misfolded protein clients (step 4) and enhances the ability of R-HSPA5 to activate SQSTM1 (step 5). Our earlier work [30] has shown that the Nt-Arg of R-HSPA5 binds the ZZ domain of SQSTM1 and allosterically activates the conformation of SQSTM1, exposing PB1 and LIR domains of SQSTM1 (step 6). This enables PB1-mediated self-aggregation of SQSTM1 along with R-HSPA5 and Ub-conjugated misfolded cargoes (step 7) and LIR-mediated interaction with LC3 (step 8), facilitating the autophagic removal of cytotoxic misfolded proteins and their aggregates. In this R-HSPA5-SQSTM1 circuit, PARK7 acts as a cofactor/co-chaperone of R-HSPA5 to modulate SQSTM1-dependent macroautophagy under TNFSF10-induced stresses and possibly other types of stress as well.

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: Hypothetical model for the role of PARK7 in autophagic protein quality control. In this model, TNFSF10 causes mitochondrial misregulation and oxidative stress associated with the excessive generation of ROS. This causes the formation of cytosolic misfolded proteins that are tagged with Ub but cannot be degraded by the UPS (step 1). In response to the proteotoxicity, cells induce autophagic protein quality control, which involves the Nt-arginylation of the ER-resident HSPA5 (step 2). In parallel, PARK7 is oxidized (step 3). The resulting PARK7 binds R-HSPA5 as its cofactor/co-chaperone that facilitates the ability of association with binding Ub-tagged misfolded protein clients (step 4) and enhances the ability of R-HSPA5 to activate SQSTM1 (step 5). Our earlier work [30] has shown that the Nt-Arg of R-HSPA5 binds the ZZ domain of SQSTM1 and allosterically activates the conformation of SQSTM1, exposing PB1 and LIR domains of SQSTM1 (step 6). This enables PB1-mediated self-aggregation of SQSTM1 along with R-HSPA5 and Ub-conjugated misfolded cargoes (step 7) and LIR-mediated interaction with LC3 (step 8), facilitating the autophagic removal of cytotoxic misfolded proteins and their aggregates. In this R-HSPA5-SQSTM1 circuit, PARK7 acts as a cofactor/co-chaperone of R-HSPA5 to modulate SQSTM1-dependent macroautophagy under TNFSF10-induced stresses and possibly other types of stress as well.

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Binding Assay

    PARK7 is oxidized by TNFSF10 and the oxidation of PARK7 is required for the interaction of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h, followed by immunoblotting with specific antibodies for oxidized PARK7 and PARK7. (b) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h or co-treated with 10 ng/ml TNFSF10 and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (c) HCT116 cells were treated with 250 μM tert-butyl hydroperoxide (tBHP) for 3 h or co-treated with 250 μM tBHP and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (d) HCT116 cells were co-transfected with plasmids encoding Ub-R-HSPA5-GFP and one of the following: FLAG-tagged wild-type PARK7 or its C46A, C53A, and C106A mutants. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody. (e) HCT116 cells were transfected with a plasmid encoding FLAG, FLAG-PARK7, or FLAG-PARK7 C106A . After 48 h, cells were treated with 20 ng/ml TNFSF10 for 2 h. Cell lysates were immunoprecipitated with the anti-FLAG antibody and then immunoblotted with the indicated antibodies.

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: PARK7 is oxidized by TNFSF10 and the oxidation of PARK7 is required for the interaction of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h, followed by immunoblotting with specific antibodies for oxidized PARK7 and PARK7. (b) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h or co-treated with 10 ng/ml TNFSF10 and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (c) HCT116 cells were treated with 250 μM tert-butyl hydroperoxide (tBHP) for 3 h or co-treated with 250 μM tBHP and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (d) HCT116 cells were co-transfected with plasmids encoding Ub-R-HSPA5-GFP and one of the following: FLAG-tagged wild-type PARK7 or its C46A, C53A, and C106A mutants. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody. (e) HCT116 cells were transfected with a plasmid encoding FLAG, FLAG-PARK7, or FLAG-PARK7 C106A . After 48 h, cells were treated with 20 ng/ml TNFSF10 for 2 h. Cell lysates were immunoprecipitated with the anti-FLAG antibody and then immunoblotted with the indicated antibodies.

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Transfection, Immunoprecipitation, Plasmid Preparation

    TNFSF10 induces the interaction of oxidized PARK7 with R-HSPA5. (a) HCT116 cells were transfected with a plasmid encoding FLAG or FLAG-PARK7. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. (b) HCT116 cells stably expressing FLAG or FLAG-PARK7 were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were incubated with GST-PARK7 proteins for 2 h then immunoprecipitated with glutathione bead, followed by immunoblotting with the indicated antibodies. (c) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (d) HCT116 cells were treated with 200 nM bafilomycin A 1 (Baf) for 6 h or cultured in the presence of 200 nM bafilomycin A 1 for 2 h then additionally treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (e) A schematic diagram indicating the interaction between arginylated HSPA5, oxidized PARK7 (oxPARK7), and activated SQSTM1. In this mechanism, HSPA5, PARK7 and SQSTM1 are each modified, and the modification of these 3 components constitutes the complex of 3-way interaction. (f) A schematic diagram in which recombinant Ub-R/V-HSPA5-GFP proteins are processed by a deubiquitination enzyme (DUB). In this mechanism, Ub-R/V-HSPA5-GFP is expressed in HCT116 cells and its ubiquitin is cleaved by DUB and arginine or valine is exposed as a result of cleavage as shown. (g) HCT116 cells were co-transfected with plasmids encoding FLAG-PARK7 and Ub-R-HSPA5-GFP or Ub-V- HSPA5-GFP. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody.

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: TNFSF10 induces the interaction of oxidized PARK7 with R-HSPA5. (a) HCT116 cells were transfected with a plasmid encoding FLAG or FLAG-PARK7. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. (b) HCT116 cells stably expressing FLAG or FLAG-PARK7 were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were incubated with GST-PARK7 proteins for 2 h then immunoprecipitated with glutathione bead, followed by immunoblotting with the indicated antibodies. (c) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (d) HCT116 cells were treated with 200 nM bafilomycin A 1 (Baf) for 6 h or cultured in the presence of 200 nM bafilomycin A 1 for 2 h then additionally treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (e) A schematic diagram indicating the interaction between arginylated HSPA5, oxidized PARK7 (oxPARK7), and activated SQSTM1. In this mechanism, HSPA5, PARK7 and SQSTM1 are each modified, and the modification of these 3 components constitutes the complex of 3-way interaction. (f) A schematic diagram in which recombinant Ub-R/V-HSPA5-GFP proteins are processed by a deubiquitination enzyme (DUB). In this mechanism, Ub-R/V-HSPA5-GFP is expressed in HCT116 cells and its ubiquitin is cleaved by DUB and arginine or valine is exposed as a result of cleavage as shown. (g) HCT116 cells were co-transfected with plasmids encoding FLAG-PARK7 and Ub-R-HSPA5-GFP or Ub-V- HSPA5-GFP. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody.

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing, Incubation, Cell Culture, Modification, Recombinant

    PARK7 is required for the autophagic targeting of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) Cells were treated with 200 nM bafilomycin A 1 for 6 h or 10 ng/ml TNFSF10 for 4 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A 1 for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. Immunostaining analysis was performed using antibodies to R-HSPA5 (red) and SQSTM1 (green), followed by confocal microscopy. Scale bar: 10 μm. (b) HCT116 cells stably expressing scrambled shRNA or sh PARK7 were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A 1 (Baf) for 2 h and additionally treated with 10 ng/ml TNFSF10 for 4 h as described in A. (c) Quantification of R-HSPA5 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (*** p

    Journal: Autophagy

    Article Title: PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

    doi: 10.1080/15548627.2018.1491212

    Figure Lengend Snippet: PARK7 is required for the autophagic targeting of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) Cells were treated with 200 nM bafilomycin A 1 for 6 h or 10 ng/ml TNFSF10 for 4 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A 1 for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. Immunostaining analysis was performed using antibodies to R-HSPA5 (red) and SQSTM1 (green), followed by confocal microscopy. Scale bar: 10 μm. (b) HCT116 cells stably expressing scrambled shRNA or sh PARK7 were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A 1 (Baf) for 2 h and additionally treated with 10 ng/ml TNFSF10 for 4 h as described in A. (c) Quantification of R-HSPA5 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (*** p

    Article Snippet: The following primary antibodies were used: anti-PARK7(Abcam, ab76008; Cell Signaling Technology, 5933; Santa Cruz Biotechnology, sc-55,572); anti-MAP1LC3A/B (MBL, M152-3; Sigma, L7543); anti-SQSTM1 (Abcam, ab56416, ab91526; Santa Cruz Biotechnology, sc-25,575); anti-HSPA5 (Abcam, ab21685; Cell Signaling Technology, 3183); anti-WIPI2 (Abcam ab105459); anti-FK2 (Enzo Life Sciences, BML-PW8810); anti-GFP (Cell Signaling Technology, 2555); anti-FLAG (Cell Signaling Technology, 2368; Sigma, F1804); anti-HA (Cell Signaling Technology, 2367); anti-VDAC (Cell Signaling Technology, 4866); anti-CANX (Cell Signaling Technology, 2433); anti-PARP1 (Cell Signaling Technology, 9542); anti-ACTB (MP Biomedicals, 0869100; Sigma, A1978).

    Techniques: Cell Culture, Immunostaining, Confocal Microscopy, Stable Transfection, Expressing, shRNA

    Cellular processing of α-Gal mutants. A , immunostaining of wild type and mutated α-Gal transiently expressed in HeLa cells and co-staining of cellular compartment markers: BiP (ER) Giantin (Golgi), and LAMP1 (lysosomes). α-Gal

    Journal: The Journal of Biological Chemistry

    Article Title: ?-Galactosidase Aggregation Is a Determinant of Pharmacological Chaperone Efficacy on Fabry Disease Mutants *

    doi: 10.1074/jbc.M112.351056

    Figure Lengend Snippet: Cellular processing of α-Gal mutants. A , immunostaining of wild type and mutated α-Gal transiently expressed in HeLa cells and co-staining of cellular compartment markers: BiP (ER) Giantin (Golgi), and LAMP1 (lysosomes). α-Gal

    Article Snippet: The primary antibodies for the Myc tag (Invitrogen), LAMP1 for lysosomal staining (Abcam), BiP for ER staining (Abcam), and Giantin for Golgi staining (Abcam) were diluted 1:200 in blocking buffer and incubated 1 h at room temperature (or overnight at 4 °C).

    Techniques: Immunostaining, Staining

    a BrdU labelling of control primary chondrocytes and chondrocytes treated with tunicamycin with and without exogenous MANF ( n = 2). b Western blot and densitometry measurement of GRP78 levels in untreated and tunicamycin-treated primary chondrocytes with and without exogenous MANF ( n = 2). Tun tunicamycin. *** P

    Journal: Cell Stress & Chaperones

    Article Title: Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis

    doi: 10.1007/s12192-018-0953-7

    Figure Lengend Snippet: a BrdU labelling of control primary chondrocytes and chondrocytes treated with tunicamycin with and without exogenous MANF ( n = 2). b Western blot and densitometry measurement of GRP78 levels in untreated and tunicamycin-treated primary chondrocytes with and without exogenous MANF ( n = 2). Tun tunicamycin. *** P

    Article Snippet: However, in vitro treatment of Matn3 V194D chondrocytes with exogenous MANF had no effect on the amount or localisation of retained mutant matrilin-3 despite lowering the levels of GRP78 by 25% (Fig. b–d).

    Techniques: Western Blot

    a X-ray radiographs of Manf fl/fl Col2Cre − Matn3 +/+ , Manf fl/fl Col2Cre − Matn3 V194D/V194D and Manf fl/fl Col2Cre + Matn3 V194D/V194D mice at 6 weeks, showing the dramatic effect of removal of MANF from cartilage of the mouse model of MED. b Western blot and densitometry measurement of intracellular GRP78 levels in Matn3 V194D/V194D primary chondrocytes with and without exogenous MANF ( n = 4). c Western blot and densitometry measurement of intracellular matrilin-3 levels in Matn3 V194D/V194D primary chondrocytes with and without exogenous MANF ( n = 4). d Immunocytochemistry for matrilin-3 showing that exogenous MANF has no effect on the intracellular retention of mutant matrilin-3 (in green) in Matn3 V194D/V194D primary chondrocytes. DAPI was used as a counterstain. Scale bars 5 mm ( a ) and 200 μm ( d ) (colour figure online)

    Journal: Cell Stress & Chaperones

    Article Title: Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis

    doi: 10.1007/s12192-018-0953-7

    Figure Lengend Snippet: a X-ray radiographs of Manf fl/fl Col2Cre − Matn3 +/+ , Manf fl/fl Col2Cre − Matn3 V194D/V194D and Manf fl/fl Col2Cre + Matn3 V194D/V194D mice at 6 weeks, showing the dramatic effect of removal of MANF from cartilage of the mouse model of MED. b Western blot and densitometry measurement of intracellular GRP78 levels in Matn3 V194D/V194D primary chondrocytes with and without exogenous MANF ( n = 4). c Western blot and densitometry measurement of intracellular matrilin-3 levels in Matn3 V194D/V194D primary chondrocytes with and without exogenous MANF ( n = 4). d Immunocytochemistry for matrilin-3 showing that exogenous MANF has no effect on the intracellular retention of mutant matrilin-3 (in green) in Matn3 V194D/V194D primary chondrocytes. DAPI was used as a counterstain. Scale bars 5 mm ( a ) and 200 μm ( d ) (colour figure online)

    Article Snippet: However, in vitro treatment of Matn3 V194D chondrocytes with exogenous MANF had no effect on the amount or localisation of retained mutant matrilin-3 despite lowering the levels of GRP78 by 25% (Fig. b–d).

    Techniques: Mouse Assay, Western Blot, Immunocytochemistry, Mutagenesis

    a STRING network showing the known interactions between the proteins encoded by the genes differentially expressed in the Manf fl/fl Col2Cre − and Col2Cre + cartilage at P5. b Deletion of MANF increases the levels of GRP78 but not GRP94 in cartilage at P5, as shown by Western blotting and densitometry measurement ( n = 3). c Western blotting of proteins co-immunoprecipitated in the 293 cells expressing FLAG-tagged recombinant MANF

    Journal: Cell Stress & Chaperones

    Article Title: Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis

    doi: 10.1007/s12192-018-0953-7

    Figure Lengend Snippet: a STRING network showing the known interactions between the proteins encoded by the genes differentially expressed in the Manf fl/fl Col2Cre − and Col2Cre + cartilage at P5. b Deletion of MANF increases the levels of GRP78 but not GRP94 in cartilage at P5, as shown by Western blotting and densitometry measurement ( n = 3). c Western blotting of proteins co-immunoprecipitated in the 293 cells expressing FLAG-tagged recombinant MANF

    Article Snippet: However, in vitro treatment of Matn3 V194D chondrocytes with exogenous MANF had no effect on the amount or localisation of retained mutant matrilin-3 despite lowering the levels of GRP78 by 25% (Fig. b–d).

    Techniques: Western Blot, Immunoprecipitation, Expressing, Recombinant

    Schematic showing the effect of store-operated Ca 2+ entry deficiency in enamel. ( A ) WT maturation-stage ameloblast showing a well-developed ruffled border (RB) at the apical end where the Na + /Ca 2+ /K + exchanger NCKX4 is localized. In these cells, depletion of endoplasmic reticulum (ER) Ca 2+ stores results in the activation of the ER Ca 2+ sensors STIM1 and STIM2 resulting in sustained Ca 2+ entry via Orai1 and refilling of ER stores. Normal Ca 2+ homeostasis and the Ca 2+ extrusion system critically contribute to the proper mineralization of enamel. ( B ) In STIM1/STIM2–deficient cells, depletion of ER Ca 2+ stores does not result in appropriate activation of STIM1 and STIM2, and hence Ca 2+ entry via Orai1 is impaired, resulting in Ca 2+ -induced ER stress. The subsequent activation of the unfolded protein response (UPR) mechanism helps mediate this cell stress and can prevent cell death. In enamel cells deficient of STIM1 and STIM2, UPR-associated genes Bip/Grp78 and RCAN1 are upregulated. CHOP is also upregulated, which can affect the glutathione system (GSH) affecting normal S-glutathionylation of actin, preventing the normal development of the RB. Mitochondria become mislocalized as a result. Mitochondria morphology is disrupted, possibly associated with RCAN1 upregulation or with a decreased glutathione system, leading to increased ROS and abnormal mitochondrial bioenergetics. Dysfunction of STIM1 and STIM2 results in severe enamel hypomineralization and softer enamel, which increases tooth wear.

    Journal: JCI Insight

    Article Title: Store-operated Ca2+ entry controls ameloblast cell function and enamel development

    doi: 10.1172/jci.insight.91166

    Figure Lengend Snippet: Schematic showing the effect of store-operated Ca 2+ entry deficiency in enamel. ( A ) WT maturation-stage ameloblast showing a well-developed ruffled border (RB) at the apical end where the Na + /Ca 2+ /K + exchanger NCKX4 is localized. In these cells, depletion of endoplasmic reticulum (ER) Ca 2+ stores results in the activation of the ER Ca 2+ sensors STIM1 and STIM2 resulting in sustained Ca 2+ entry via Orai1 and refilling of ER stores. Normal Ca 2+ homeostasis and the Ca 2+ extrusion system critically contribute to the proper mineralization of enamel. ( B ) In STIM1/STIM2–deficient cells, depletion of ER Ca 2+ stores does not result in appropriate activation of STIM1 and STIM2, and hence Ca 2+ entry via Orai1 is impaired, resulting in Ca 2+ -induced ER stress. The subsequent activation of the unfolded protein response (UPR) mechanism helps mediate this cell stress and can prevent cell death. In enamel cells deficient of STIM1 and STIM2, UPR-associated genes Bip/Grp78 and RCAN1 are upregulated. CHOP is also upregulated, which can affect the glutathione system (GSH) affecting normal S-glutathionylation of actin, preventing the normal development of the RB. Mitochondria become mislocalized as a result. Mitochondria morphology is disrupted, possibly associated with RCAN1 upregulation or with a decreased glutathione system, leading to increased ROS and abnormal mitochondrial bioenergetics. Dysfunction of STIM1 and STIM2 results in severe enamel hypomineralization and softer enamel, which increases tooth wear.

    Article Snippet: The following primary antibodies (all rabbit raised) were used: anti-Stim1 (1:200 dilution; Sigma-Aldrich HPA012123), anti-NCKX4 (1:500 dilution, Abcam ab136968), anti-BiP/GRP78 (1:100 dilution, Abcam ab32618), anti-TOMM20 (1:100 dilution, Abcam ab186734), anti-β-Actin (1:100 dilution, SantaCruz sc-47778), and anti-Amelx (1:50 dilution; SantaCruz; sc-32892).

    Techniques: Activation Assay

    Dynamics of GRP78/BiP expression during retinal development. GRP78/BiP protein is increased at P4 through P12 in tubby retinas compared to age-matched wt, but has significantly decreased expression at P20 through P28. (A) Data shown are representative

    Journal: Experimental eye research

    Article Title: Correlation of ER stress and retinal degeneration in tubby mice

    doi: 10.1016/j.exer.2015.08.022

    Figure Lengend Snippet: Dynamics of GRP78/BiP expression during retinal development. GRP78/BiP protein is increased at P4 through P12 in tubby retinas compared to age-matched wt, but has significantly decreased expression at P20 through P28. (A) Data shown are representative

    Article Snippet: The slides were blocked with 5% BSA, then incubated in the following primary antibodies for 2 hrs at room temperature: mouse anti-rhodopsin (R1D4) (1:2000, generous gift from Dr. Robert Molday, Columbia University, Vancouver, Canada), rabbit anti-M-opsin (1:500, Millipore, AB5405) and rabbit anti-GRP78 BiP (1:300, Abcam, ab21685).

    Techniques: Expressing

    Distribution and alteration of GRP78/BiP protein during retinal development. Immunocytochemistry showed that in wt and tubby retinas, GRP78/BiP is mainly expressed in the RPE, INL and GCL at P4 through P12 with expression being stronger in tubby retinas

    Journal: Experimental eye research

    Article Title: Correlation of ER stress and retinal degeneration in tubby mice

    doi: 10.1016/j.exer.2015.08.022

    Figure Lengend Snippet: Distribution and alteration of GRP78/BiP protein during retinal development. Immunocytochemistry showed that in wt and tubby retinas, GRP78/BiP is mainly expressed in the RPE, INL and GCL at P4 through P12 with expression being stronger in tubby retinas

    Article Snippet: The slides were blocked with 5% BSA, then incubated in the following primary antibodies for 2 hrs at room temperature: mouse anti-rhodopsin (R1D4) (1:2000, generous gift from Dr. Robert Molday, Columbia University, Vancouver, Canada), rabbit anti-M-opsin (1:500, Millipore, AB5405) and rabbit anti-GRP78 BiP (1:300, Abcam, ab21685).

    Techniques: Immunocytochemistry, Expressing

    Murine and rat IGSF1-2 proteins do not traffic to the plasma membrane of transfected cells. A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with streptavidin-HRP (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.

    Journal: PLoS ONE

    Article Title: The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein

    doi: 10.1371/journal.pone.0180731

    Figure Lengend Snippet: Murine and rat IGSF1-2 proteins do not traffic to the plasma membrane of transfected cells. A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with streptavidin-HRP (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.

    Article Snippet: Double-labeling for Myc and GRP-78/BiP (Abcam, ab21685; 1:500) was performed similarly with a few exceptions: 1) cells were seeded at a density of 5x104 , 2) fixation in 4% paraformaldehyde was reduced to 2 min, 3) incubation in primary antibodies was for 1 h at room temperature rather than overnight, and 4) the secondary antibodies were Alexa Fluor 488 donkey anti-mouse (A-21202, ThermoFisher Scientific; 1:500) and Alexa Fluor 594 donkey anti-rabbit (A-21207, ThermoFisher Scientific; 1:500).

    Techniques: Transfection, Expressing, IA, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Cell Culture, Immunofluorescence, Staining, Confocal Microscopy

    Anti-GRP78 antibody inhibits TMUV infection in BHK-21 cells. BHK-21 cells were pre-incubated with 100 μg/ml rabbit IgG, anti-GRP78 N-terminal or anti-GRP78 C-terminal antibodies at 4°C or 1 h followed by TMUV infection. The level of TMUV RNA in rabbit IgG incubated cells was taken as 1 for determining the relative RNA levels. The viral RNA was compared to those in rabbit IgG incubated cells. Data were presented from three independent experiments and statistic analysis was done with SPSS software. The asterisk designates statistically significant differences ( p

    Journal: Frontiers in Microbiology

    Article Title: Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection

    doi: 10.3389/fmicb.2018.00694

    Figure Lengend Snippet: Anti-GRP78 antibody inhibits TMUV infection in BHK-21 cells. BHK-21 cells were pre-incubated with 100 μg/ml rabbit IgG, anti-GRP78 N-terminal or anti-GRP78 C-terminal antibodies at 4°C or 1 h followed by TMUV infection. The level of TMUV RNA in rabbit IgG incubated cells was taken as 1 for determining the relative RNA levels. The viral RNA was compared to those in rabbit IgG incubated cells. Data were presented from three independent experiments and statistic analysis was done with SPSS software. The asterisk designates statistically significant differences ( p

    Article Snippet: GRP78 N-terminal (ab32618, rabbit), GRP78 C-terminal (ab21685, rabbit), goat anti-rabbit IgG (Alexa Fluor 488, ab150077), goat anti-mouse (Alexa Fluor 594, ab150080), and anti-GAPDH antibody (ab8245) were purchased from Abcam.

    Techniques: Infection, Incubation, Software

    Immunofluorescence studies of the TFR2 p.Gly792Arg mutant in human cell lines. (A) C-terminal FLAG wild-type or mutated p.Gly792Arg TFR2 constructs were transiently transfected and visualized using anti-FLAG antibody (red) in an epifluorescence microscope in permeabilized and nonper-meabilized Huh7cells. E-Cadherin was used as a membrane protein control (green). DAPI (blue) was used to detected DNA and visualize nuclear morphology. (B) N-terminal FLAG wild-type or mutated p.Gly792Arg TFR2 constructs were transiently transfected as above and visualized in permeabilized HeLa cells, using anti-FLAG (red) and anti-GRP78-BiP (green, ER marker) antibodies. Note that G792R=Gly792Arg.

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3

    doi: 10.1002/mgg3.136

    Figure Lengend Snippet: Immunofluorescence studies of the TFR2 p.Gly792Arg mutant in human cell lines. (A) C-terminal FLAG wild-type or mutated p.Gly792Arg TFR2 constructs were transiently transfected and visualized using anti-FLAG antibody (red) in an epifluorescence microscope in permeabilized and nonper-meabilized Huh7cells. E-Cadherin was used as a membrane protein control (green). DAPI (blue) was used to detected DNA and visualize nuclear morphology. (B) N-terminal FLAG wild-type or mutated p.Gly792Arg TFR2 constructs were transiently transfected as above and visualized in permeabilized HeLa cells, using anti-FLAG (red) and anti-GRP78-BiP (green, ER marker) antibodies. Note that G792R=Gly792Arg.

    Article Snippet: At 48 h posttransfection, cells were fixed with 4% paraformaldehyde (Sigma Aldrich), permeabilized or not with Triton X-100 0.1%; incubated with the appropriate antibodies (mouse monoclonal anti-FLAG, [Sigma], rat monoclonal anti-E-cadherin [Millipore, CA, USA] or rabbit polyclonal anti-GRP78 BiP [Abcam, Cambridge, UK] as primary; AlexaFluor 488 rabbit-anti-rat, AlexaFluor 488 goat-anti-rabbit and AlexaFluor 568 goat-anti-mouse [Invitrogen Molecular Probes, OR, USA] as secondary); washed with PBS-Tween 0.02% and PBS and mounted in DAPI-containing mounting medium (Vector Laboratories, CA, USA).

    Techniques: Immunofluorescence, Mutagenesis, Construct, Transfection, Microscopy, Marker

    TGEV infection activates all three UPR signaling pathways in vivo . Twelve 2-day-old SPF piglets were orally inoculated with TGEV strain H87 or with DMEM as uninfected controls. All the piglets were euthanized by the end of the study, which was terminated at 48 hpi. (A) Virus replication in the ileum was tested by qPCR. (B) Representative microphotographs of viral antigen immunochemical staining in TGEV-noninfected and -infected ileum tissues (magnification, ×200). (C) GRP78 expression in ileum tissues was detected by qPCR. (D) Protein levels of GRP78, ATF6, p-PERK, PERK, p-eIF2α, and eIF2α in ileum samples from TGEV-noninfected and -infected piglets. (E) XBP1s/XBP1t (XBP1-spliced/XBP1-total) ratio and mRNA expression of the ERdj4 gene in ileum samples. (F) XBP1 mRNA splicing in ileum samples from TGEV-noninfected and -infected piglets. XBP1s, spliced XBP1; XBP1u, unspliced XBP1. Means and SD of the results from three independent experiments are shown. * , P

    Journal: Journal of Virology

    Article Title: The PERK Arm of the Unfolded Protein Response Negatively Regulates Transmissible Gastroenteritis Virus Replication by Suppressing Protein Translation and Promoting Type I Interferon Production

    doi: 10.1128/JVI.00431-18

    Figure Lengend Snippet: TGEV infection activates all three UPR signaling pathways in vivo . Twelve 2-day-old SPF piglets were orally inoculated with TGEV strain H87 or with DMEM as uninfected controls. All the piglets were euthanized by the end of the study, which was terminated at 48 hpi. (A) Virus replication in the ileum was tested by qPCR. (B) Representative microphotographs of viral antigen immunochemical staining in TGEV-noninfected and -infected ileum tissues (magnification, ×200). (C) GRP78 expression in ileum tissues was detected by qPCR. (D) Protein levels of GRP78, ATF6, p-PERK, PERK, p-eIF2α, and eIF2α in ileum samples from TGEV-noninfected and -infected piglets. (E) XBP1s/XBP1t (XBP1-spliced/XBP1-total) ratio and mRNA expression of the ERdj4 gene in ileum samples. (F) XBP1 mRNA splicing in ileum samples from TGEV-noninfected and -infected piglets. XBP1s, spliced XBP1; XBP1u, unspliced XBP1. Means and SD of the results from three independent experiments are shown. * , P

    Article Snippet: Antibodies against GRP78 (ab21685), p-PERK (ab192591), p-eIF2α (Ser51) (ab32157), ATF6 (ab122897), ATF4 (ab1371), and β-actin (ab6276) were purchased from Abcam (Cambridge, MA).

    Techniques: Infection, In Vivo, Real-time Polymerase Chain Reaction, Staining, Expressing

    TGEV infection induces ER stress in ST and IPEC-J2 cells. ST cells and IPEC-J2 cells were infected with TGEV H87 at an MOI of 1; samples were collected at 0, 6, 12, 24, 36, and 48 hpi. (A) One-step growth curve of TGEV in ST cells and IPEC-J2 cells. (B) A time-dependent increase of GRP78 expression was revealed by qPCR in ST cells and IPEC-J2 cells. Total RNA was isolated, and the transcriptional levels of GRP78 were measured by qPCR at different time points (0 to 48 h) after infection. (C) Elevated protein expression of GRP78 was confirmed by Western blotting in ST cells and IPEC-J2 cells. Tu (2 μg/ml) was used as a positive control for UPR activation; β-actin was used as a loading control. (D) Relative GRP78 levels. (E) TGEV-induced UPR was dependent on active viral replication. ST cells were infected with UV-inactivated TGEV or treated with Tu (2 μg/ml). The GRP78 expression in ST cells at different time points was determined by Western blotting using anti-GRP78 antibody. (F) Relative GRP78 levels. Means and SD of the results from three independent experiments are shown.

    Journal: Journal of Virology

    Article Title: The PERK Arm of the Unfolded Protein Response Negatively Regulates Transmissible Gastroenteritis Virus Replication by Suppressing Protein Translation and Promoting Type I Interferon Production

    doi: 10.1128/JVI.00431-18

    Figure Lengend Snippet: TGEV infection induces ER stress in ST and IPEC-J2 cells. ST cells and IPEC-J2 cells were infected with TGEV H87 at an MOI of 1; samples were collected at 0, 6, 12, 24, 36, and 48 hpi. (A) One-step growth curve of TGEV in ST cells and IPEC-J2 cells. (B) A time-dependent increase of GRP78 expression was revealed by qPCR in ST cells and IPEC-J2 cells. Total RNA was isolated, and the transcriptional levels of GRP78 were measured by qPCR at different time points (0 to 48 h) after infection. (C) Elevated protein expression of GRP78 was confirmed by Western blotting in ST cells and IPEC-J2 cells. Tu (2 μg/ml) was used as a positive control for UPR activation; β-actin was used as a loading control. (D) Relative GRP78 levels. (E) TGEV-induced UPR was dependent on active viral replication. ST cells were infected with UV-inactivated TGEV or treated with Tu (2 μg/ml). The GRP78 expression in ST cells at different time points was determined by Western blotting using anti-GRP78 antibody. (F) Relative GRP78 levels. Means and SD of the results from three independent experiments are shown.

    Article Snippet: Antibodies against GRP78 (ab21685), p-PERK (ab192591), p-eIF2α (Ser51) (ab32157), ATF6 (ab122897), ATF4 (ab1371), and β-actin (ab6276) were purchased from Abcam (Cambridge, MA).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Positive Control, Activation Assay

    Antibodies against MHC I inhibit JEV infection. Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or MHC-I antibody alone or in combination with GRP78 C- and N-terminus-specific rabbit antibodies on ice for 1 h, followed by

    Journal: Journal of Virology

    Article Title: GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

    doi: 10.1128/JVI.02274-16

    Figure Lengend Snippet: Antibodies against MHC I inhibit JEV infection. Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or MHC-I antibody alone or in combination with GRP78 C- and N-terminus-specific rabbit antibodies on ice for 1 h, followed by

    Article Snippet: Antibodies against JEV E protein (ab41671, mouse; ab81193, mouse; ab26950, rabbit), GRP78 N terminus (ab32618, rabbit), GRP78 C terminus (ab21685, rabbit), MHC-I (ab52922, rabbit), and calreticulin (ab22683, mouse) were purchased from Abcam, while antibody against GAPDH (2118S, mouse) was purchased from Cell Signaling Technology.

    Techniques: Infection, Incubation

    Role of GRP78 in binding and entry of JEV in neuronal cells. (A) Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific antibodies on ice for 1 h, followed by the addition of DiI-labeled JEV

    Journal: Journal of Virology

    Article Title: GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

    doi: 10.1128/JVI.02274-16

    Figure Lengend Snippet: Role of GRP78 in binding and entry of JEV in neuronal cells. (A) Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific antibodies on ice for 1 h, followed by the addition of DiI-labeled JEV

    Article Snippet: Antibodies against JEV E protein (ab41671, mouse; ab81193, mouse; ab26950, rabbit), GRP78 N terminus (ab32618, rabbit), GRP78 C terminus (ab21685, rabbit), MHC-I (ab52922, rabbit), and calreticulin (ab22683, mouse) were purchased from Abcam, while antibody against GAPDH (2118S, mouse) was purchased from Cell Signaling Technology.

    Techniques: Binding Assay, Incubation, Labeling

    GRP78 N-terminal antibody inhibits JEV infection in mammalian cells. Primary mouse cortical neurons and Neuro2a and Huh-7 cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific rabbit antibodies on ice

    Journal: Journal of Virology

    Article Title: GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

    doi: 10.1128/JVI.02274-16

    Figure Lengend Snippet: GRP78 N-terminal antibody inhibits JEV infection in mammalian cells. Primary mouse cortical neurons and Neuro2a and Huh-7 cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific rabbit antibodies on ice

    Article Snippet: Antibodies against JEV E protein (ab41671, mouse; ab81193, mouse; ab26950, rabbit), GRP78 N terminus (ab32618, rabbit), GRP78 C terminus (ab21685, rabbit), MHC-I (ab52922, rabbit), and calreticulin (ab22683, mouse) were purchased from Abcam, while antibody against GAPDH (2118S, mouse) was purchased from Cell Signaling Technology.

    Techniques: Infection, Incubation

    Anti-Grp78 immunohistochemistry showing differential expression of Grp78 in the pancreas of drug treated orthotopic murine model: Treatment with gemcitabine plus paclitaxel (Chemo) or Chemo + sunitinib increases the Grp78 levels in the ductal carcinoma (arrows) and surrounding cells ( p

    Journal: Journal of Translational Medicine

    Article Title: Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

    doi: 10.1186/s12967-018-1562-z

    Figure Lengend Snippet: Anti-Grp78 immunohistochemistry showing differential expression of Grp78 in the pancreas of drug treated orthotopic murine model: Treatment with gemcitabine plus paclitaxel (Chemo) or Chemo + sunitinib increases the Grp78 levels in the ductal carcinoma (arrows) and surrounding cells ( p

    Article Snippet: GRP78 expression was analyzed by IHC using respective anti-GRP78 antibodies (Abcam) specific to mouse or human for the respective tissue sections and the intensity of immunostaining was analyzed in both ductal carcinoma and adjacent acini and surrounding inflammatory regions.

    Techniques: Immunohistochemistry, Expressing

    Anti-GRP78 immunostaining in PDAC tissue microarray and patient-derived pancreatic tissue biopsies. a Representative images of Anti-GRP78 immunohistochemistry (red chromogens) on PDAC tissue microarray showing different levels of GRP78 expression. PDAC tissues show consistently higher GRP78 expression ( arrow ) compared to non-tumor adjacent tissues (NAT). b Bar-charts showing increased level of GRP78 expression in PDAC tissues ( p

    Journal: Journal of Translational Medicine

    Article Title: Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

    doi: 10.1186/s12967-018-1562-z

    Figure Lengend Snippet: Anti-GRP78 immunostaining in PDAC tissue microarray and patient-derived pancreatic tissue biopsies. a Representative images of Anti-GRP78 immunohistochemistry (red chromogens) on PDAC tissue microarray showing different levels of GRP78 expression. PDAC tissues show consistently higher GRP78 expression ( arrow ) compared to non-tumor adjacent tissues (NAT). b Bar-charts showing increased level of GRP78 expression in PDAC tissues ( p

    Article Snippet: GRP78 expression was analyzed by IHC using respective anti-GRP78 antibodies (Abcam) specific to mouse or human for the respective tissue sections and the intensity of immunostaining was analyzed in both ductal carcinoma and adjacent acini and surrounding inflammatory regions.

    Techniques: Immunostaining, Microarray, Derivative Assay, Immunohistochemistry, Expressing

    XBP1 splicing, lysosomal activities and cell viability in human PDAC cells treated with ER stress and autophagy modulating drugs. a Human PDAC derived Panc02.03 cells were treated with 2.5 µM tunicamycin and/or 25 µM STF for 72 h, and RT-PCR was performed on the extracted mRNA of these cells to monitor the XBP1 splicing and GRP78 expression. As expected, XBP1 splicing is increased in tunicamycin treated cells compared to DMSO treated cells, while co-treatment of STF-083010 reverses the upregulated splicing to the normal splicing levels, comparable to that of DMSO treated cells. GRP78 is increased with tunicamycin treatment, but is not further altered by the addition of STF-083010 treatment. b TEM images of DMSO (left) and tunicamycin-treated (middle) and STF-083010 (right) Panc02.03 cells, showing organelle pathology of ER vesicles (arrowhead), and lysosomes (arrow). Both tunicamycin and STF-083010 treated cells exhibit enlarged ER vesicles and have increased lysosomes in the cytoplasm. Bar-charts shows significantly increased lysosome contents in STF-083010 treated cells. Scale bar, 500 nM. c Panc02.03 cells were treated with tunicamycin (2.5 µM), STF-083010 (25 µM) with or without 4-PBA (100 µM) for 72 h, and the lysosomes were viewed by fluorescent microscopy using lysotracker staining. Both STF-083010 and tunicamycin treatments led to increased lysotracker staining, compared to DMSO treatment groups. Co-treatment with 4PBA reduced the elevated lysotracker staining to a comparable level with the DMSO treated group. Chloroquine at 50 µM causes cell clumping and cell death and reduced lysotracker staining. Scale bar: 5 µm. d Bar-charts showing normalized cell viability ratio of PDAC cells treated with STF-083010 (25 µM), chloroquine (50 µM), gemcitabine (250 nM) and various combinations (n = 4). Human PDAC-derived Panc02.03 cells were treated with various concentrations of single or combination treatments for 72 h and the viability was measured by Trypan Blue exclusion assay. STF-083010 and chloroquine show additive effects with gemcitabine ( p

    Journal: Journal of Translational Medicine

    Article Title: Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

    doi: 10.1186/s12967-018-1562-z

    Figure Lengend Snippet: XBP1 splicing, lysosomal activities and cell viability in human PDAC cells treated with ER stress and autophagy modulating drugs. a Human PDAC derived Panc02.03 cells were treated with 2.5 µM tunicamycin and/or 25 µM STF for 72 h, and RT-PCR was performed on the extracted mRNA of these cells to monitor the XBP1 splicing and GRP78 expression. As expected, XBP1 splicing is increased in tunicamycin treated cells compared to DMSO treated cells, while co-treatment of STF-083010 reverses the upregulated splicing to the normal splicing levels, comparable to that of DMSO treated cells. GRP78 is increased with tunicamycin treatment, but is not further altered by the addition of STF-083010 treatment. b TEM images of DMSO (left) and tunicamycin-treated (middle) and STF-083010 (right) Panc02.03 cells, showing organelle pathology of ER vesicles (arrowhead), and lysosomes (arrow). Both tunicamycin and STF-083010 treated cells exhibit enlarged ER vesicles and have increased lysosomes in the cytoplasm. Bar-charts shows significantly increased lysosome contents in STF-083010 treated cells. Scale bar, 500 nM. c Panc02.03 cells were treated with tunicamycin (2.5 µM), STF-083010 (25 µM) with or without 4-PBA (100 µM) for 72 h, and the lysosomes were viewed by fluorescent microscopy using lysotracker staining. Both STF-083010 and tunicamycin treatments led to increased lysotracker staining, compared to DMSO treatment groups. Co-treatment with 4PBA reduced the elevated lysotracker staining to a comparable level with the DMSO treated group. Chloroquine at 50 µM causes cell clumping and cell death and reduced lysotracker staining. Scale bar: 5 µm. d Bar-charts showing normalized cell viability ratio of PDAC cells treated with STF-083010 (25 µM), chloroquine (50 µM), gemcitabine (250 nM) and various combinations (n = 4). Human PDAC-derived Panc02.03 cells were treated with various concentrations of single or combination treatments for 72 h and the viability was measured by Trypan Blue exclusion assay. STF-083010 and chloroquine show additive effects with gemcitabine ( p

    Article Snippet: GRP78 expression was analyzed by IHC using respective anti-GRP78 antibodies (Abcam) specific to mouse or human for the respective tissue sections and the intensity of immunostaining was analyzed in both ductal carcinoma and adjacent acini and surrounding inflammatory regions.

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transmission Electron Microscopy, Microscopy, Staining, Trypan Blue Exclusion Assay

    Markers of endoplasmic reticulum stress in the renal cortex. Western blot analysis of A, grp-78 and B, calnexin in the renal cortex of wild type and Itga8 -/- mice after induction of unilateral ureter obstruction (UUO). Exemplary western blots are shown from the UUO groups. Itga8 +/+, wild type mice; Itga8 -/-, Itga8 -deficient mice. Data are means±SEM.

    Journal: PLoS ONE

    Article Title: Alpha8 Integrin (Itga8) Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover

    doi: 10.1371/journal.pone.0150471

    Figure Lengend Snippet: Markers of endoplasmic reticulum stress in the renal cortex. Western blot analysis of A, grp-78 and B, calnexin in the renal cortex of wild type and Itga8 -/- mice after induction of unilateral ureter obstruction (UUO). Exemplary western blots are shown from the UUO groups. Itga8 +/+, wild type mice; Itga8 -/-, Itga8 -deficient mice. Data are means±SEM.

    Article Snippet: Antibodies for Western blot analysis The primary antibody to survivin (AF886; R & D Systems, Wiesbaden, Germany) was used at a dilution of 1:400, to grp-78 (ab21685; Abcam, Cambridge, UK) at a dilution of 1:200, to calnexin (ab75801; Abcam) at a dilution of 1:1500 and to p-SMAD2/3 (SC-11769; Santa Cruz) at a dilution of 1:5000.

    Techniques: Western Blot, Mouse Assay

    Aminoglycosides induce the UPR. ( a–c ) qPCR analysis. HEK wild-type cells were treated with geneticin (16 μ M) or gentamicin (400 μ M) and incubated for the indicated times. Expression of mRNA for CHOP ( a ), BiP ( b ) and GRP94 ( c ) is shown. Means+S.D. of fold induction are presented relative to 0 h (untreated) sample ( n =3); * P

    Journal: Cell Death & Disease

    Article Title: XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death

    doi: 10.1038/cddis.2015.108

    Figure Lengend Snippet: Aminoglycosides induce the UPR. ( a–c ) qPCR analysis. HEK wild-type cells were treated with geneticin (16 μ M) or gentamicin (400 μ M) and incubated for the indicated times. Expression of mRNA for CHOP ( a ), BiP ( b ) and GRP94 ( c ) is shown. Means+S.D. of fold induction are presented relative to 0 h (untreated) sample ( n =3); * P

    Article Snippet: The specific antibodies used in this study were: anti-BiP antibody (Abcam, ab21685); anti-GRP94 antibody (Abcam, ab87886); anti-ATF4 antibody (Abcam, ab23760); anti-β -actin antibody (A1978-200UL; Sigma-Aldrich); HRP-conjugated goat anti-rabbit (Invitrogen, G-21234) and goat anti-mouse antibodies (Invitrogen, A10551).

    Techniques: Real-time Polymerase Chain Reaction, Incubation, Expressing

    Silencing of PDIA6 modulates IRE1α activity (A), (B). NIH-3T3 cells were transfected with XBP1 splicing reporter and siRNA directed against PDIA6, BiP, IRE1α or negative scrambled control (Neg) and treated with thapsigargin (Thap) (A,B) or tunicamycin (Tun) (B). In (A)* P-value = 0.002598. In (B) * P-value = 0.002598. NS, not significant. (C) RT-PCR analysis of XBP1 splicing in control cells and cells treated with siRNA against PDIA6. Thap, thapsigargin. Right panel, PCR products were digested with Pst1. XBP1s, spliced XBP1. Neg, negative scrambled control. (D), (E) Q-PCR analysis of total XBP1 (D) and spliced XBP1 (E) in control and PDIA6 silenced cells treated with thapsigargin (Thap). P-value = 0.0124. NS, not significant. Neg, negative scrambled control. (G). UPRE splicing reporter activity in NIH-3T3 fibroblasts transfected with PDIA6 siRNA. (H). ERSE splicing reporter activity in NIH-3T3 fibroblasts transfected with PDIA6 siRNA. *P-value = 0.004284. (I). NIH-3T3 fibroblasts were transfected with PDIA6 siRNA followed by treatment with thapsigargin (Thap). * P -value = 0.0098. All data in the figure are representative of more than 3 biological replicates.

    Journal: Science signaling

    Article Title: Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

    doi: 10.1126/scisignal.2004983

    Figure Lengend Snippet: Silencing of PDIA6 modulates IRE1α activity (A), (B). NIH-3T3 cells were transfected with XBP1 splicing reporter and siRNA directed against PDIA6, BiP, IRE1α or negative scrambled control (Neg) and treated with thapsigargin (Thap) (A,B) or tunicamycin (Tun) (B). In (A)* P-value = 0.002598. In (B) * P-value = 0.002598. NS, not significant. (C) RT-PCR analysis of XBP1 splicing in control cells and cells treated with siRNA against PDIA6. Thap, thapsigargin. Right panel, PCR products were digested with Pst1. XBP1s, spliced XBP1. Neg, negative scrambled control. (D), (E) Q-PCR analysis of total XBP1 (D) and spliced XBP1 (E) in control and PDIA6 silenced cells treated with thapsigargin (Thap). P-value = 0.0124. NS, not significant. Neg, negative scrambled control. (G). UPRE splicing reporter activity in NIH-3T3 fibroblasts transfected with PDIA6 siRNA. (H). ERSE splicing reporter activity in NIH-3T3 fibroblasts transfected with PDIA6 siRNA. *P-value = 0.004284. (I). NIH-3T3 fibroblasts were transfected with PDIA6 siRNA followed by treatment with thapsigargin (Thap). * P -value = 0.0098. All data in the figure are representative of more than 3 biological replicates.

    Article Snippet: Antibodies used were rabbit anti-PDIA6 (Abcam, ab11432), rabbit anti-BiP (Abcam, ab21685), rabbit anti-IRE1α (Abcam, ab37073), goat anti-calreticulin, rabbit anti-GAPDH (Abcam, ab9483) and rabbit anti-tubulin antibodies (Abcam, ab6046).

    Techniques: Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    PDIA6 interacts with BiP and IRE1α and controls IRE1α activity (A to C). Top blots: To detect associations of IRE1 with proteins involved in ER stress responses, immunoprecipitations (IP) using anti-BiP, anti-PDIA6, or anti-IRE-1α were performed in COS-1 cells expressing IRE1-NLD and immunoblotted with anti-IRE1α antibodies. Middle and bottom blots: immunoprecipitations (IP) using anti-BiP, anti-PDIA6, or anti-IRE-1α were performed in NIH-3T3 fibroblasts and immunoblotted with anti-PDIA6 or anti-BiP. *, depicts the location of IRE1-NLD protein band;

    Journal: Science signaling

    Article Title: Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

    doi: 10.1126/scisignal.2004983

    Figure Lengend Snippet: PDIA6 interacts with BiP and IRE1α and controls IRE1α activity (A to C). Top blots: To detect associations of IRE1 with proteins involved in ER stress responses, immunoprecipitations (IP) using anti-BiP, anti-PDIA6, or anti-IRE-1α were performed in COS-1 cells expressing IRE1-NLD and immunoblotted with anti-IRE1α antibodies. Middle and bottom blots: immunoprecipitations (IP) using anti-BiP, anti-PDIA6, or anti-IRE-1α were performed in NIH-3T3 fibroblasts and immunoblotted with anti-PDIA6 or anti-BiP. *, depicts the location of IRE1-NLD protein band;

    Article Snippet: Antibodies used were rabbit anti-PDIA6 (Abcam, ab11432), rabbit anti-BiP (Abcam, ab21685), rabbit anti-IRE1α (Abcam, ab37073), goat anti-calreticulin, rabbit anti-GAPDH (Abcam, ab9483) and rabbit anti-tubulin antibodies (Abcam, ab6046).

    Techniques: Activity Assay, Expressing

    Organization of secretory pathway organelles in Russell body-positive cells. Fluorescent micrographs of HEK293 cells transfected with N35W variant LC construct. On day-2 post transfection, suspension cultured cells were seeded onto poly-lysine coated glass coverslips and statically incubated for 24 hr. On day-3, cells were fixed, permeabilized, and co-stained with Texas Red-conjugated anti-kappa chain polyclonal antibody and specific antibodies against various organelle markers. (A, B) ER markers BiP and calnexin. (C) A cis-/medial-Golgi marker giantin. (D) A trans-Golgi marker p230. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. In panel C top row, arrowhead points to a normal ribbon-like Golgi morphology in a non-transfected cell. Unlabeled scale bar represents 10 μm.

    Journal: mAbs

    Article Title: Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic

    doi: 10.1080/19420862.2017.1314875

    Figure Lengend Snippet: Organization of secretory pathway organelles in Russell body-positive cells. Fluorescent micrographs of HEK293 cells transfected with N35W variant LC construct. On day-2 post transfection, suspension cultured cells were seeded onto poly-lysine coated glass coverslips and statically incubated for 24 hr. On day-3, cells were fixed, permeabilized, and co-stained with Texas Red-conjugated anti-kappa chain polyclonal antibody and specific antibodies against various organelle markers. (A, B) ER markers BiP and calnexin. (C) A cis-/medial-Golgi marker giantin. (D) A trans-Golgi marker p230. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. In panel C top row, arrowhead points to a normal ribbon-like Golgi morphology in a non-transfected cell. Unlabeled scale bar represents 10 μm.

    Article Snippet: Rabbit polyclonal anti-BiP (cat. ab21685, lot –1), mouse monoclonal anti-CD27L (BU69) (cat. ab77868), and rabbit polyclonal anti-phospho-eIF2α (Ser51) (cat. ab4837, lot –1) were from abcam.

    Techniques: Transfection, Variant Assay, Construct, Cell Culture, Incubation, Staining, Marker

    Positions of Grp78/BiP positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm

    Journal: BMC Veterinary Research

    Article Title: Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

    doi: 10.1186/s12917-018-1748-x

    Figure Lengend Snippet: Positions of Grp78/BiP positive staining cells in lamellar tissue. All images are from laminitic samples. Grp78/BiP (green) and WGA (red) were localized as described in Methods. Images were selected to show the range of Grp78/BiP staining patterns observed in laminitic tissues. In the Abaxial region, Grp78/BiP expressing cells (yellow arrows) are found in multiple layers of suprabasal cells, or limited to a single layer of cells adjacent to the keratinized axis. In the middle region, Grp78/BiP positive cells (yellow arrows) are found adjacent to the keratinized axis (KA), often several cell layers deep. In some cases, positive cells are found along the length of the keratinized axis, while in other tissues expression is limited to groups of cells. All images are shown at the same magnification. Scale bar is 50 μm

    Article Snippet: Paraffin-embedded 6 μm thick sections of formalin-fixed tissues were used for immunofluorescent localization of Grp78/BiP (Cell Signaling Technology clone C50B12).

    Techniques: Staining, Whole Genome Amplification, Expressing

    Grp78/BiP is expressed at higher levels in laminitic tissues. a . Representative Grp78/BiP immunoblot, reprobed for alpha-tubulin as a loading control. A band of ~ 78 kd co-migrates with the Hela (human) protein. b : Legend to Figure S1A)). Lanes in (A,B) are labeled as: control (C), EL front limb (F) and EL hind limb (H). ( c ). Box plot of normalized Grp78/BiP band intensities in control ( n = 6), EL front limbs (=12) and EL hind limbs ( n = 8). Horizontal lines represent the median value for each group. Grp78/BiP expression was not significantly different between controls and EL hind limbs ( p = 0.77) d . Comparison of Grp78/BiP expression in paired samples ( n = 8) from EL front and hind limbs from the same horses. The inset shows the 4 sample pairs with small differences in Grp78/BiP band intensities between EL front and hind limb samples. The same pattern is apparent in 3 of 4 pairs, where the value for Grp78/BiP level in the front limb is greater than that in the hind limb

    Journal: BMC Veterinary Research

    Article Title: Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

    doi: 10.1186/s12917-018-1748-x

    Figure Lengend Snippet: Grp78/BiP is expressed at higher levels in laminitic tissues. a . Representative Grp78/BiP immunoblot, reprobed for alpha-tubulin as a loading control. A band of ~ 78 kd co-migrates with the Hela (human) protein. b : Legend to Figure S1A)). Lanes in (A,B) are labeled as: control (C), EL front limb (F) and EL hind limb (H). ( c ). Box plot of normalized Grp78/BiP band intensities in control ( n = 6), EL front limbs (=12) and EL hind limbs ( n = 8). Horizontal lines represent the median value for each group. Grp78/BiP expression was not significantly different between controls and EL hind limbs ( p = 0.77) d . Comparison of Grp78/BiP expression in paired samples ( n = 8) from EL front and hind limbs from the same horses. The inset shows the 4 sample pairs with small differences in Grp78/BiP band intensities between EL front and hind limb samples. The same pattern is apparent in 3 of 4 pairs, where the value for Grp78/BiP level in the front limb is greater than that in the hind limb

    Article Snippet: Paraffin-embedded 6 μm thick sections of formalin-fixed tissues were used for immunofluorescent localization of Grp78/BiP (Cell Signaling Technology clone C50B12).

    Techniques: Labeling, Expressing

    Localization of Grp78/BiP to suprabasal keratinocytes of the epidermal lamellae in laminitic tissue. Tissue sections were stained as described in Methods to localize Grp78/BiP (green) and wheat germ agglutinin (WGA; red) to outline cell plasma membranes (epidermis) and extracellular matrix (dermis). The Grp78/BiP channel is also shown separately in grayscale for each image. Representative images from Abaxial (AbAx), Middle (Mid) and Axial positions (Ax) are shown for control and laminitic samples. The control tissues showed no significant Grp78/BiP staining under the conditions used. In contrast, laminitic tissues showed bright Grp78/BiP expression in suprabasal epidermal keratinocytes located at the abaxial region and along the keratinized axis. Positive staining was absent from SELs in the axial region. All images are shown at the same magnification. Scale bar is 50 μm

    Journal: BMC Veterinary Research

    Article Title: Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

    doi: 10.1186/s12917-018-1748-x

    Figure Lengend Snippet: Localization of Grp78/BiP to suprabasal keratinocytes of the epidermal lamellae in laminitic tissue. Tissue sections were stained as described in Methods to localize Grp78/BiP (green) and wheat germ agglutinin (WGA; red) to outline cell plasma membranes (epidermis) and extracellular matrix (dermis). The Grp78/BiP channel is also shown separately in grayscale for each image. Representative images from Abaxial (AbAx), Middle (Mid) and Axial positions (Ax) are shown for control and laminitic samples. The control tissues showed no significant Grp78/BiP staining under the conditions used. In contrast, laminitic tissues showed bright Grp78/BiP expression in suprabasal epidermal keratinocytes located at the abaxial region and along the keratinized axis. Positive staining was absent from SELs in the axial region. All images are shown at the same magnification. Scale bar is 50 μm

    Article Snippet: Paraffin-embedded 6 μm thick sections of formalin-fixed tissues were used for immunofluorescent localization of Grp78/BiP (Cell Signaling Technology clone C50B12).

    Techniques: Staining, Whole Genome Amplification, Expressing

    High fat and ethanol diets activate PERK signaling in pancreatic acinar cells. ( A ) Western blotting analysis for peIF2α, BiP/GRP78 (upper band) or β-actin (as a control) in pancreatic tissue of WT or Mist1 −/− mice fed breeder's chow (BC) or LDC-E or LDC-HF diets for 6 weeks. Quantitative analysis of peIF2α ( B ) or BiP/GRP78 ( C ) accumulation from (A) relative to β-actin accumulation. LDC-E and LDC-HF diets lead to significant increases in peIF2α compared to BC diets in WT mice but not in Mist1 −/− tissue. Conversely, while BiP/GRP78 levels are unchanged in WT mice, they decrease upon exposure to LDC diets in Mist1 −/− mice. Groups were compared using One way ANOVA and a Tukey post-hoc tests: *P

    Journal: PLoS ONE

    Article Title: The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

    doi: 10.1371/journal.pone.0028863

    Figure Lengend Snippet: High fat and ethanol diets activate PERK signaling in pancreatic acinar cells. ( A ) Western blotting analysis for peIF2α, BiP/GRP78 (upper band) or β-actin (as a control) in pancreatic tissue of WT or Mist1 −/− mice fed breeder's chow (BC) or LDC-E or LDC-HF diets for 6 weeks. Quantitative analysis of peIF2α ( B ) or BiP/GRP78 ( C ) accumulation from (A) relative to β-actin accumulation. LDC-E and LDC-HF diets lead to significant increases in peIF2α compared to BC diets in WT mice but not in Mist1 −/− tissue. Conversely, while BiP/GRP78 levels are unchanged in WT mice, they decrease upon exposure to LDC diets in Mist1 −/− mice. Groups were compared using One way ANOVA and a Tukey post-hoc tests: *P

    Article Snippet: Another target of ATF6α, BiP/GRP78 also decreased in LDC-E and LDC-HF fed Mist1−/− mice , supporting the findings that Mist1−/− mice had a reduction in ATF6 signaling as a result of ethanol or high-fat consumption.

    Techniques: Western Blot, Mouse Assay

    Mist1 −/− pancreatic tissue exhibits increased activation of the UPR. ( A ) Western blot analysis of key UPR markers in 2 month old WT and Mist1 −/− whole pancreatic lysates. Mist1 −/− extracts show significantly increased accumulations of BiP/GRP78 ( B ), GADD34 ( C ) and sXBP1 ( D ), but not peIF2α (p = 0.743) or uXBP1 (p = 0.532) relative to WT pancreatic tissue. *P

    Journal: PLoS ONE

    Article Title: The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

    doi: 10.1371/journal.pone.0028863

    Figure Lengend Snippet: Mist1 −/− pancreatic tissue exhibits increased activation of the UPR. ( A ) Western blot analysis of key UPR markers in 2 month old WT and Mist1 −/− whole pancreatic lysates. Mist1 −/− extracts show significantly increased accumulations of BiP/GRP78 ( B ), GADD34 ( C ) and sXBP1 ( D ), but not peIF2α (p = 0.743) or uXBP1 (p = 0.532) relative to WT pancreatic tissue. *P

    Article Snippet: Another target of ATF6α, BiP/GRP78 also decreased in LDC-E and LDC-HF fed Mist1−/− mice , supporting the findings that Mist1−/− mice had a reduction in ATF6 signaling as a result of ethanol or high-fat consumption.

    Techniques: Activation Assay, Western Blot

    Dexamethasone treatment reduces incidence of pseudoimplantation and NF κ B-mediated inflammation in Msx1/Msx2 d/d uteri under delayed conditions. A , incidence of pseudoimplantation sites ( pseudoIS ) in vehicle- ( Veh ), celecoxib- ( Cele ), or dexamethasone-treated ( Dex ) Msx1/Msx2 d/d uteri under delayed conditions. Bars showing color-coded segments represent the percentage of mice with pseudoIS ( red ), blue bands ( green ), and recovered blastocysts ( blue ). Although percentages of pseudoIS with faint or no blue bands at the site of blastocysts are shown within parentheses in the red segments of bars , numbers of mice used in each set of experiments are shown above the bars. B , representative images of Msx1/Msx2 d/d uteri after vehicle or Dex treatment as compared with dexamethasone-treated floxed uteri. Arrow , faint blue band. C , left panel , Western blotting results showing reduced levels of pIκB, α-subunits of the 20S proteasome, p-eIF2α, and HSPA5/Bip in Msx1/Msx2 d/d mice after dexamethasone treatment. Right panels , quantification of Western blotting results (means ± S.E.). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus *

    doi: 10.1074/jbc.M115.655001

    Figure Lengend Snippet: Dexamethasone treatment reduces incidence of pseudoimplantation and NF κ B-mediated inflammation in Msx1/Msx2 d/d uteri under delayed conditions. A , incidence of pseudoimplantation sites ( pseudoIS ) in vehicle- ( Veh ), celecoxib- ( Cele ), or dexamethasone-treated ( Dex ) Msx1/Msx2 d/d uteri under delayed conditions. Bars showing color-coded segments represent the percentage of mice with pseudoIS ( red ), blue bands ( green ), and recovered blastocysts ( blue ). Although percentages of pseudoIS with faint or no blue bands at the site of blastocysts are shown within parentheses in the red segments of bars , numbers of mice used in each set of experiments are shown above the bars. B , representative images of Msx1/Msx2 d/d uteri after vehicle or Dex treatment as compared with dexamethasone-treated floxed uteri. Arrow , faint blue band. C , left panel , Western blotting results showing reduced levels of pIκB, α-subunits of the 20S proteasome, p-eIF2α, and HSPA5/Bip in Msx1/Msx2 d/d mice after dexamethasone treatment. Right panels , quantification of Western blotting results (means ± S.E.). *, p

    Article Snippet: Antibodies to phosphorylated IκB (mouse, Cell Signaling Technology, 9246), total IκB (rabbit, Cell Signaling Technology, 9242), 20S proteasome α-1, 2, 3, 5, 6, 7 subunits (mouse, Enzo, MCP231), α6 (rabbit, laboratory-generated), Rpt6 (mouse, laboratory-generated), Rpn8 (rabbit, laboratory-generated), HSPA5/Bip (goat, sc-1050), phosphorylated eIF2α (rabbit, Cell Signaling, 3597S), total eIF2α (rabbit, sc-11386), ubiquitin lysine 48 (rabbit, Millipore, 05-1307), and ubiquitin lysine 63 (rabbit, Millipore, 05-1308) were used as previously described ( ).

    Techniques: Mouse Assay, Western Blot

    ER stress is evident in Msx1/Msx2 d/d uteri under delayed conditions and is aggravated by bortezomib treatment. A , representative results of immunofluorescence showing apical epithelial expression of HSPA5/Bip in Msx1/Msx2 f/f uteri, with increased intensity and dispersed Bip-positive stromal cells in Msx1/Msx2 d/d uteri. Stromal Bip expression is widespread at the pseudoimplantation site ( Pseudo-IS ). Arrowheads , dispersed Bip-positive cells. Bar , 250 μm. B , blastocysts recovery ( top bar graph ) from Msx1/Msx2 d/d after bortezomib treatment females was significantly lower with enhanced rate of pseudoimplantation sites ( bottom bar graph ) compared with floxed littermates on day 10 (means ± S.E.). Numbers in parentheses indicate number of females that produced blastocysts or pseudoimplantation sites, respectively, compared with the number of females assessed.

    Journal: The Journal of Biological Chemistry

    Article Title: Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus *

    doi: 10.1074/jbc.M115.655001

    Figure Lengend Snippet: ER stress is evident in Msx1/Msx2 d/d uteri under delayed conditions and is aggravated by bortezomib treatment. A , representative results of immunofluorescence showing apical epithelial expression of HSPA5/Bip in Msx1/Msx2 f/f uteri, with increased intensity and dispersed Bip-positive stromal cells in Msx1/Msx2 d/d uteri. Stromal Bip expression is widespread at the pseudoimplantation site ( Pseudo-IS ). Arrowheads , dispersed Bip-positive cells. Bar , 250 μm. B , blastocysts recovery ( top bar graph ) from Msx1/Msx2 d/d after bortezomib treatment females was significantly lower with enhanced rate of pseudoimplantation sites ( bottom bar graph ) compared with floxed littermates on day 10 (means ± S.E.). Numbers in parentheses indicate number of females that produced blastocysts or pseudoimplantation sites, respectively, compared with the number of females assessed.

    Article Snippet: Antibodies to phosphorylated IκB (mouse, Cell Signaling Technology, 9246), total IκB (rabbit, Cell Signaling Technology, 9242), 20S proteasome α-1, 2, 3, 5, 6, 7 subunits (mouse, Enzo, MCP231), α6 (rabbit, laboratory-generated), Rpt6 (mouse, laboratory-generated), Rpn8 (rabbit, laboratory-generated), HSPA5/Bip (goat, sc-1050), phosphorylated eIF2α (rabbit, Cell Signaling, 3597S), total eIF2α (rabbit, sc-11386), ubiquitin lysine 48 (rabbit, Millipore, 05-1307), and ubiquitin lysine 63 (rabbit, Millipore, 05-1308) were used as previously described ( ).

    Techniques: Immunofluorescence, Expressing, Produced

    Effects of HFD on markers of adipocyte function and injury in WT and NOX2KO mice. Expression of ( A ) PPARγ, ( B ) adiponectin, ( C ) GADD153/CHOP, and ( D ) GRP78 were evaluated in tissue homogenates prepared from epididymal adipose depots. Data were

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: NOX2 deficiency attenuates markers of adiposopathy and brain injury induced by high-fat diet

    doi: 10.1152/ajpendo.00398.2012

    Figure Lengend Snippet: Effects of HFD on markers of adipocyte function and injury in WT and NOX2KO mice. Expression of ( A ) PPARγ, ( B ) adiponectin, ( C ) GADD153/CHOP, and ( D ) GRP78 were evaluated in tissue homogenates prepared from epididymal adipose depots. Data were

    Article Snippet: Blots prepared from adipose tissue were processed using anti-Iba-1 (1:500, Wako Chemicals, Richmond, VA), anti-PPARγ1/2 (1:1,000, Abcam, Cambridge, MA), anti-adiponectin (1:1,000, Abcam), anti-GADD153/CHOP (1:5,000, Abcam), anti-GRP78 (1:500, Novus Biologicals), and anti-tubulin (1:1,000, Wako Chemicals).

    Techniques: Mouse Assay, Expressing

    Microscopic analysis of the distribution of BiP/GRP78 and CHOP/Gadd153 in immunohistochemically stained cochleae (SP, ×400). (a-1–a-3) Cells that were positively stained for BiP/GRP were located in the organ of Corti, the lateral wall, and the spiral ganglion, respectively, in the 1d group (positive expression: immuoreactivity is brown versus the blue hematoxylin stain, indicated by ↑). (b-1–b-3) Cells that were positively stained for CHOP/Gadd153 were located in the same regions as those shown in (a), in the 1d group (positive expression: immunoreactivity is tan or medium-brown particles versus hematoxylin-stain, indicated by ↑). (c-1–c-3) Negative control incubated with PBS instead of the primary antibodies (organ of Corti, the lateral wall, and the spiral ganglion, respectively)

    Journal: Noise & Health

    Article Title: The Protective Effect of the Endoplasmic Reticulum Stress-Related Factors BiP/GRP78 and CHOP/Gadd153 on Noise-induced Hearing Loss in Guinea Pigs

    doi: 10.4103/1463-1741.192481

    Figure Lengend Snippet: Microscopic analysis of the distribution of BiP/GRP78 and CHOP/Gadd153 in immunohistochemically stained cochleae (SP, ×400). (a-1–a-3) Cells that were positively stained for BiP/GRP were located in the organ of Corti, the lateral wall, and the spiral ganglion, respectively, in the 1d group (positive expression: immuoreactivity is brown versus the blue hematoxylin stain, indicated by ↑). (b-1–b-3) Cells that were positively stained for CHOP/Gadd153 were located in the same regions as those shown in (a), in the 1d group (positive expression: immunoreactivity is tan or medium-brown particles versus hematoxylin-stain, indicated by ↑). (c-1–c-3) Negative control incubated with PBS instead of the primary antibodies (organ of Corti, the lateral wall, and the spiral ganglion, respectively)

    Article Snippet: BiP/GRP78 and CHOP/Gadd153 Western blot analysis: Higher levels of expression in the experimental groups The results of the Western blot analysis examining the levels of BiP/GRP78 and CHOP/Gadd153 protein are shown in .

    Techniques: Staining, Expressing, Negative Control, Incubation

    Expression of BiP/GRP78 and CHOP/Gadd153 proteins evaluated by Western blot analysis (a). Relative protein level of BiP/GRP 78 and CHOP/Gadd153 was analyzed by using one-way ANOVA (b, c). *Comparison with control group, P

    Journal: Noise & Health

    Article Title: The Protective Effect of the Endoplasmic Reticulum Stress-Related Factors BiP/GRP78 and CHOP/Gadd153 on Noise-induced Hearing Loss in Guinea Pigs

    doi: 10.4103/1463-1741.192481

    Figure Lengend Snippet: Expression of BiP/GRP78 and CHOP/Gadd153 proteins evaluated by Western blot analysis (a). Relative protein level of BiP/GRP 78 and CHOP/Gadd153 was analyzed by using one-way ANOVA (b, c). *Comparison with control group, P

    Article Snippet: BiP/GRP78 and CHOP/Gadd153 Western blot analysis: Higher levels of expression in the experimental groups The results of the Western blot analysis examining the levels of BiP/GRP78 and CHOP/Gadd153 protein are shown in .

    Techniques: Expressing, Western Blot

    Grp78/BiP protein identification and quantitation using label-free spectral counting-based mass spectrometry. (A) Workflow of label-free spectral counting-based protein quantitative analysis using LC-MS/MS. Protein samples were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and gel pieces excised at ~80 kDa for the purpose of in-gel trypsin digestion and LC-MS/MS analysis. Protein quantitative data analysis was conducted using spectral counting and interpreted by normalized total spectra numbers. (B) Grp78/BiP protein identification and peptide coverage using LC-MS/MS. (C) Representative mass-to-charge ratio spectrum and b-/y ions fragmentation of Grp78/BiP peptide (highlighted in B ). (D) Protein quantitative data analysis using the ratio of normalized total spectra numbers of Grp78/BiP to a house keeping protein GAPDH. Three independent muscles from 120–140d old wild type (WT) and ALS-Tg mice were analyzed. * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice

    doi: 10.3389/fncel.2015.00170

    Figure Lengend Snippet: Grp78/BiP protein identification and quantitation using label-free spectral counting-based mass spectrometry. (A) Workflow of label-free spectral counting-based protein quantitative analysis using LC-MS/MS. Protein samples were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and gel pieces excised at ~80 kDa for the purpose of in-gel trypsin digestion and LC-MS/MS analysis. Protein quantitative data analysis was conducted using spectral counting and interpreted by normalized total spectra numbers. (B) Grp78/BiP protein identification and peptide coverage using LC-MS/MS. (C) Representative mass-to-charge ratio spectrum and b-/y ions fragmentation of Grp78/BiP peptide (highlighted in B ). (D) Protein quantitative data analysis using the ratio of normalized total spectra numbers of Grp78/BiP to a house keeping protein GAPDH. Three independent muscles from 120–140d old wild type (WT) and ALS-Tg mice were analyzed. * p

    Article Snippet: Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature.

    Techniques: Quantitation Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Polyacrylamide Gel Electrophoresis, Mouse Assay

    Grp78/BiP and CHOP protein levels in cardiac muscle and liver tissue of G93A*SOD1 ALS-Tg mice. (A) Cardiac muscle (HRT) was collected and protein levels determined using western blot technique. Grp78/BiP and CHOP antibodies were used and three postnatal ages were examined: early pre-symptomatic (70d; n = 3 each for WT and ALS-Tg), late pre-symptomatic (90d; n = 5 each for WT and ALS-Tg), and symptomatic (120–140d; n = 3 each for WT and ALS-Tg) mice. (B) Liver tissues (LIV) was collected and protein levels were determined as described above. Analysis of average arbitrary units (AU) obtained by densitometry of Grp78/BiP and CHOP in HRT (C) and in LIV (D) . Data in (C,D) are presented as mean ± S.E.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice

    doi: 10.3389/fncel.2015.00170

    Figure Lengend Snippet: Grp78/BiP and CHOP protein levels in cardiac muscle and liver tissue of G93A*SOD1 ALS-Tg mice. (A) Cardiac muscle (HRT) was collected and protein levels determined using western blot technique. Grp78/BiP and CHOP antibodies were used and three postnatal ages were examined: early pre-symptomatic (70d; n = 3 each for WT and ALS-Tg), late pre-symptomatic (90d; n = 5 each for WT and ALS-Tg), and symptomatic (120–140d; n = 3 each for WT and ALS-Tg) mice. (B) Liver tissues (LIV) was collected and protein levels were determined as described above. Analysis of average arbitrary units (AU) obtained by densitometry of Grp78/BiP and CHOP in HRT (C) and in LIV (D) . Data in (C,D) are presented as mean ± S.E.

    Article Snippet: Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature.

    Techniques: Mouse Assay, Western Blot

    Comparison of Grp78/BiP and CHOP protein levels between white and red gastrocnemius (RG) muscle tissues of G93A*SOD1 ALS-Tg mice. (A) Image of deep portion of gastrocnemius muscle showing white and RG (yellow dashed areas) muscle region. (B) White (WG) and red (RG) gastrocnemius muscle tissues of symptomatic animals (120–140d; n = 3 each for WT and ALS-Tg) were collected. Grp78/BiP and CHOP protein levels were determined using western blot technique. (C) Analysis of Grp78/BiP and CHOP average ratio of ALS-Tg to WT. Data in (B) is presented as mean ± S.E; *, p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice

    doi: 10.3389/fncel.2015.00170

    Figure Lengend Snippet: Comparison of Grp78/BiP and CHOP protein levels between white and red gastrocnemius (RG) muscle tissues of G93A*SOD1 ALS-Tg mice. (A) Image of deep portion of gastrocnemius muscle showing white and RG (yellow dashed areas) muscle region. (B) White (WG) and red (RG) gastrocnemius muscle tissues of symptomatic animals (120–140d; n = 3 each for WT and ALS-Tg) were collected. Grp78/BiP and CHOP protein levels were determined using western blot technique. (C) Analysis of Grp78/BiP and CHOP average ratio of ALS-Tg to WT. Data in (B) is presented as mean ± S.E; *, p

    Article Snippet: Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature.

    Techniques: Mouse Assay, Western Blot

    Schematic figure showing unfolded protein response and endoplasmic reticulum (ER) stress pathway and its proposed role in skeletal muscle atrophy and weakness in ALS . In skeletal muscle of ALS-Tg mice, the G93A*SOD1 mutation leads to oxidative stress and protein misfolding. This leads to an age-dependent activation of ER stress sensors protein kinase RNA-activated-like ER kinase (PERK) and inositol-requiring kinase 1-alpha (IRE1α). Normally, these ER stress sensors physically interact with the ER chaperone immunoglobulin binding protein (Grp78/BiP) which suppresses their activation but accumulation of unfolded/misfolded proteins activates Grp78/BiP, including an upregulation of Grp78/BiP and PDI protein expression. Prolonged and severe ER stress can trigger apoptosis by ER stress-specific cell death signals, including C/EBP homologous protein (CHOP) and caspase-12, leading to muscle atrophy.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice

    doi: 10.3389/fncel.2015.00170

    Figure Lengend Snippet: Schematic figure showing unfolded protein response and endoplasmic reticulum (ER) stress pathway and its proposed role in skeletal muscle atrophy and weakness in ALS . In skeletal muscle of ALS-Tg mice, the G93A*SOD1 mutation leads to oxidative stress and protein misfolding. This leads to an age-dependent activation of ER stress sensors protein kinase RNA-activated-like ER kinase (PERK) and inositol-requiring kinase 1-alpha (IRE1α). Normally, these ER stress sensors physically interact with the ER chaperone immunoglobulin binding protein (Grp78/BiP) which suppresses their activation but accumulation of unfolded/misfolded proteins activates Grp78/BiP, including an upregulation of Grp78/BiP and PDI protein expression. Prolonged and severe ER stress can trigger apoptosis by ER stress-specific cell death signals, including C/EBP homologous protein (CHOP) and caspase-12, leading to muscle atrophy.

    Article Snippet: Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature.

    Techniques: Mouse Assay, Mutagenesis, Activation Assay, Binding Assay, Expressing

    ER chaperones Grp78/BiP and protein disulfide isomerase (PDI) are up-regulated in skeletal muscle of G93A*SOD1 ALS-Tg mice. (A) WG muscle tissues were used to determine Grp78/BiP and PDI expressions using western blot technique from different ages of wild-type (WT) and transgenic G93A*SOD1 (ALS-Tg) mice. Representative images of Grp78/BiP and PDI are shown. Three postnatal ages were examined as follows: early pre-symptomatic (70d; n = 3 each for WT and ALS-Tg), late pre-symptomatic (90d; n = 5 each for WT and ALS-Tg), and symptomatic (120–140d; n = 3 each for WT and ALS-Tg) mice. (B,C) Analysis of average arbitrary units (AU) obtained by densitometry of PDI. Data in B are presented as mean ± S.E; **, p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice

    doi: 10.3389/fncel.2015.00170

    Figure Lengend Snippet: ER chaperones Grp78/BiP and protein disulfide isomerase (PDI) are up-regulated in skeletal muscle of G93A*SOD1 ALS-Tg mice. (A) WG muscle tissues were used to determine Grp78/BiP and PDI expressions using western blot technique from different ages of wild-type (WT) and transgenic G93A*SOD1 (ALS-Tg) mice. Representative images of Grp78/BiP and PDI are shown. Three postnatal ages were examined as follows: early pre-symptomatic (70d; n = 3 each for WT and ALS-Tg), late pre-symptomatic (90d; n = 5 each for WT and ALS-Tg), and symptomatic (120–140d; n = 3 each for WT and ALS-Tg) mice. (B,C) Analysis of average arbitrary units (AU) obtained by densitometry of PDI. Data in B are presented as mean ± S.E; **, p

    Article Snippet: Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature.

    Techniques: Mouse Assay, Western Blot, Transgenic Assay

    MSA induces the UPR in human melanoma. ( a ) MSA treatment (15 μM) initiates the unfolded protein response in SK-Mel 28 melanoma cells as shown by increased levels of Bip/GRP78. ( b) MSA activates IRE1 as assessed by analysis of XBP1 cDNA. In the mature message produced by IRE1 the PST 1 cleavage site is spliced out giving the 447 bp product. ( c ) elF2α is phosphorylated by release of PERK from the ER of melanoma cells treated with 15 μM MSA for the indicated times. Results are representative of 2 separate experiments.

    Journal: Nutrients

    Article Title: Selenium for the Prevention of Cutaneous Melanoma

    doi: 10.3390/nu5030725

    Figure Lengend Snippet: MSA induces the UPR in human melanoma. ( a ) MSA treatment (15 μM) initiates the unfolded protein response in SK-Mel 28 melanoma cells as shown by increased levels of Bip/GRP78. ( b) MSA activates IRE1 as assessed by analysis of XBP1 cDNA. In the mature message produced by IRE1 the PST 1 cleavage site is spliced out giving the 447 bp product. ( c ) elF2α is phosphorylated by release of PERK from the ER of melanoma cells treated with 15 μM MSA for the indicated times. Results are representative of 2 separate experiments.

    Article Snippet: The Bip/GRP78 antibody was purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: Produced

    Deletion of Tlr2 inhibits increased cytokine sensitivity in Trim58 -deficient myeloid cells. ( A ) Assessment of TLR2, mature IL-1β, arginase-1, and BIP/GRP78 protein synthesis in peritoneal myeloid (PM) cells from male Trim58 +/+ and Trim58 −/− mice ( n = 4–6 per genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by immunoblot (IB) analysis. ( B ) Assessment of mature IL-1β protein synthesis in PM cells from male Tlr2 −/− , Trim58 −/− and Trim58 −/− Tlr2 −/− mice ( n = 4–6/genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by IB analysis. Blots were reprobed with anti-GAPDH to confirm equal loading. Right margin, molecular size markers (kDa). Representative results of at least two independent experiments are shown.

    Journal: The Journal of Immunology Author Choice

    Article Title: TRIM58 Restrains Intestinal Mucosal Inflammation by Negatively Regulating TLR2 in Myeloid Cells

    doi: 10.4049/jimmunol.1900413

    Figure Lengend Snippet: Deletion of Tlr2 inhibits increased cytokine sensitivity in Trim58 -deficient myeloid cells. ( A ) Assessment of TLR2, mature IL-1β, arginase-1, and BIP/GRP78 protein synthesis in peritoneal myeloid (PM) cells from male Trim58 +/+ and Trim58 −/− mice ( n = 4–6 per genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by immunoblot (IB) analysis. ( B ) Assessment of mature IL-1β protein synthesis in PM cells from male Tlr2 −/− , Trim58 −/− and Trim58 −/− Tlr2 −/− mice ( n = 4–6/genotype) stimulated with TNF-α (100 ng/ml) and IFN-γ (5 ng/ml) for 24.5 h by IB analysis. Blots were reprobed with anti-GAPDH to confirm equal loading. Right margin, molecular size markers (kDa). Representative results of at least two independent experiments are shown.

    Article Snippet: CD3e (SP7) Ab was from Thermo Fisher Scientific, BIP/GRP78 (40/BiP) from BD Biosciences, IL-1β from Abcam (catalog no. ab9722), and HA (H6908) was from Merck.

    Techniques: Mouse Assay

    MCD medium-induced ER stress activation in McA cells. Total cell lysate (70 μg of protein) was analyzed by immunoblotting for phospho-JNK, phospho-PERK, phospho-eIF2α, CHOP, BiP/GRP78, and GAPDH. A , McA cells were treated as described

    Journal: The Journal of Biological Chemistry

    Article Title: PKC? Is Activated in a Dietary Model of Steatohepatitis and Regulates Endoplasmic Reticulum Stress and Cell Death *

    doi: 10.1074/jbc.M110.168575

    Figure Lengend Snippet: MCD medium-induced ER stress activation in McA cells. Total cell lysate (70 μg of protein) was analyzed by immunoblotting for phospho-JNK, phospho-PERK, phospho-eIF2α, CHOP, BiP/GRP78, and GAPDH. A , McA cells were treated as described

    Article Snippet: Monoclonal antibodies to α-tubulin and BiP/GRP78 were from Sigma-Aldrich and BD Biosciences, respectively.

    Techniques: Activation Assay

    ERp72 and GRP78/BiP protein are elevated and ERp72 oxidized state is increased in AdDNPerk 832/13 beta cells . A. Triplicate samples were probed with antibodies to ERp72, ERp57, BiP and actin, which served as the loading control. B. ERp72 expression was increased in islets from P2 Perk KO mice. Samples are from duplicate mice for each genotype. C. At 24 hr or 36 hr post-transduction, protein samples were isolated and treated with AMS to differentiate the reduced and oxidized forms of ERp72 and ERp57 on PAGE gels. Western blots showed increased oxidized isoforms of both ERp72 and ERp57 in 832/13 cells infected with AdDNPerk compared to cells infected with AdLacZ .

    Journal: BMC Cell Biology

    Article Title: Acute ablation of PERK results in ER dysfunctions followed by reduced insulin secretion and cell proliferation

    doi: 10.1186/1471-2121-10-61

    Figure Lengend Snippet: ERp72 and GRP78/BiP protein are elevated and ERp72 oxidized state is increased in AdDNPerk 832/13 beta cells . A. Triplicate samples were probed with antibodies to ERp72, ERp57, BiP and actin, which served as the loading control. B. ERp72 expression was increased in islets from P2 Perk KO mice. Samples are from duplicate mice for each genotype. C. At 24 hr or 36 hr post-transduction, protein samples were isolated and treated with AMS to differentiate the reduced and oxidized forms of ERp72 and ERp57 on PAGE gels. Western blots showed increased oxidized isoforms of both ERp72 and ERp57 in 832/13 cells infected with AdDNPerk compared to cells infected with AdLacZ .

    Article Snippet: Primary antibodies used in the analysis were: ERp72 (1:2500, Stressgen, Inc), GRP78/BiP (1:500, Santa Cruz, Inc), ERp57 (1:300, Santa Cruz), and anti-GFP (Sigma, Inc.) to detect EFYP-ATF6.

    Techniques: Expressing, Mouse Assay, Transduction, Isolation, Affinity Magnetic Separation, Polyacrylamide Gel Electrophoresis, Western Blot, Infection

    Retinal ER stress and UPR evaluation in Wfs1 −/− mice. Immunoblots (A) detected GRP78/BiP, PDI, IRE1, and beta actin in protein lysates of 12 month old Wfs1 +/+ (n = 3) and Wfs1 −/− (n = 3) mouse retinas and in mouse NIH3T3 fibroblasts treated with thapsigargin. Mean relative quantities for each protein according to Wfs1 genotype were obtained after normalization with beta actin values. Significance (*) is indicated when p

    Journal: PLoS ONE

    Article Title: Impairment of Visual Function and Retinal ER Stress Activation in Wfs1-Deficient Mice

    doi: 10.1371/journal.pone.0097222

    Figure Lengend Snippet: Retinal ER stress and UPR evaluation in Wfs1 −/− mice. Immunoblots (A) detected GRP78/BiP, PDI, IRE1, and beta actin in protein lysates of 12 month old Wfs1 +/+ (n = 3) and Wfs1 −/− (n = 3) mouse retinas and in mouse NIH3T3 fibroblasts treated with thapsigargin. Mean relative quantities for each protein according to Wfs1 genotype were obtained after normalization with beta actin values. Significance (*) is indicated when p

    Article Snippet: Primary antibodies used in our experiments were anti-beta actin (Sigma, Lyon, France), anti-GRP78/BiP (Abcam, Paris, France), anti-PDI (Cell Signaling Technologies, Danvers, MA), anti-Ire1α (CST), PERK (Cell Signaling, Danvers, USA), Phospho_PERK (Santa Cruz Biotechnology, Heidelberg, Germany), ATF6 (ABCAM, Cambridge, UK) and LC3 (Sigma, Saint Louis, USA).

    Techniques: Mouse Assay, Western Blot

    Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Activated Clotting Time Assay, Incubation, Infection, Standard Deviation, Derivative Assay

    Identification of GRP78 as a target membrane protein of the MERS-CoV spike. A, silver staining of membrane proteins of BEAS2B cells transfected with pcDNA–MERS-CoV–S1–V5. Membrane extracts were immunoprecipitated ( IP ) with V5 antibody and Sepharose A/G beads, followed by washing and eluting with glycine ( lane 1 ). Sepharose beads were boiled in sample buffer after glycine elution ( lane 2 ). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads ( lane 3 ). B, expression of MERS-CoV–S1-V5 was detected by Western blotting ( WB ) with an anti-ERS-CoV spike antibody. C, silver staining of membrane proteins of BEAS2B cells. The membrane extracts were immunoprecipitated with purified recombinant MERS-CoV–S1–FLAG protein using anti-FLAG M2 antibody and Sepharose A/G beads, followed by washing and eluting with 3× FLAG peptides ( lane 1 ). Sepharose beads were boiled in sample buffer after 3× FLAG peptide elution ( lane 2 ). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads ( lane 3 ). D, 5 μg of sedimented membrane extracts were run on SDS-PAGE and subjected to Western blots using antibodies against the plasma membrane marker (EGFR and pan-cadherin), endoplasmic reticulum marker (calreticulin), Golgi marker (giantin), and nucleus marker (lamin A). E, gel fragment indicated by the red arrowhead in A and C was excised for LC-MS/MS analysis. MS/MS data were searched against all mammalian protein databases in NCBI and Swiss-Prot. The protein was identified as GRP78 with significant hits over different domains of the sequence.

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: Identification of GRP78 as a target membrane protein of the MERS-CoV spike. A, silver staining of membrane proteins of BEAS2B cells transfected with pcDNA–MERS-CoV–S1–V5. Membrane extracts were immunoprecipitated ( IP ) with V5 antibody and Sepharose A/G beads, followed by washing and eluting with glycine ( lane 1 ). Sepharose beads were boiled in sample buffer after glycine elution ( lane 2 ). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads ( lane 3 ). B, expression of MERS-CoV–S1-V5 was detected by Western blotting ( WB ) with an anti-ERS-CoV spike antibody. C, silver staining of membrane proteins of BEAS2B cells. The membrane extracts were immunoprecipitated with purified recombinant MERS-CoV–S1–FLAG protein using anti-FLAG M2 antibody and Sepharose A/G beads, followed by washing and eluting with 3× FLAG peptides ( lane 1 ). Sepharose beads were boiled in sample buffer after 3× FLAG peptide elution ( lane 2 ). Membrane extracts were immunoprecipitated with mouse isotype control and Sepharose A/G beads ( lane 3 ). D, 5 μg of sedimented membrane extracts were run on SDS-PAGE and subjected to Western blots using antibodies against the plasma membrane marker (EGFR and pan-cadherin), endoplasmic reticulum marker (calreticulin), Golgi marker (giantin), and nucleus marker (lamin A). E, gel fragment indicated by the red arrowhead in A and C was excised for LC-MS/MS analysis. MS/MS data were searched against all mammalian protein databases in NCBI and Swiss-Prot. The protein was identified as GRP78 with significant hits over different domains of the sequence.

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Silver Staining, Transfection, Immunoprecipitation, Expressing, Western Blot, Purification, Recombinant, SDS Page, Marker, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus ( A ), bronchiole ( B ), and alveolus ( C ). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D . Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D .

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus ( A ), bronchiole ( B ), and alveolus ( C ). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D . Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D .

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Expressing, Immunostaining, Labeling

    GRP78 is abundantly expressed on the cell surface of mammalian cells. Surface GRP78 expression was detected on mammalian cell lines with flow cytometry with no cell permeabilization. The immunostaining was performed for human lung cell lines ( A ), human extrapulmonary cell lines, human primary macrophages, and human primary T cells ( B ), as well as nonhuman cell lines ( C ). D, percentage of GRP78-positive cells quantified with DPP4 included for comparisons. E, MFI of GRP78 on the cell surface was quantified with isotype and DPP4 staining included as controls. F, sequence homology between human GRP78 and GRP78 in other mammals. Gates in A–C represented the percentage of GRP78-positive cells. Data in D and E represented mean and standard deviation from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: GRP78 is abundantly expressed on the cell surface of mammalian cells. Surface GRP78 expression was detected on mammalian cell lines with flow cytometry with no cell permeabilization. The immunostaining was performed for human lung cell lines ( A ), human extrapulmonary cell lines, human primary macrophages, and human primary T cells ( B ), as well as nonhuman cell lines ( C ). D, percentage of GRP78-positive cells quantified with DPP4 included for comparisons. E, MFI of GRP78 on the cell surface was quantified with isotype and DPP4 staining included as controls. F, sequence homology between human GRP78 and GRP78 in other mammals. Gates in A–C represented the percentage of GRP78-positive cells. Data in D and E represented mean and standard deviation from three independent experiments.

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Expressing, Flow Cytometry, Cytometry, Immunostaining, Staining, Sequencing, Standard Deviation

    GRP78 interacts with the MERS-CoV spike. A, BHK21 cells were transfected with pcDNA–GRP78–V5 ( lanes 1 and 2 ) or empty vector ( lane 3 ). The cell lysate was immunoprecipitated ( IP ) with either purified recombinant MERS-CoV–S1–FLAG protein ( lanes 1 and 3 ) or E. coli bacterial alkaline phosphatase ( BAP )-FLAG protein ( lane 2 ) pre-adsorbed onto anti-FLAG M2-agarose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-V5 antibody. B, reciprocal co-IP was performed using GRP78 as the bait protein. Purified MERS-CoV–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5 Sepharose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-GRP78 antibody. C, membrane fraction of Huh7 cells was extracted and immunoprecipitated with either MERS-CoV–S1–FLAG( lanes 1 and 3 ) or BAP–FLAG ( lane 2 ). D, reciprocal co-IP was performed using GRP78 as the bait. Mouse IgG was used in place of the membrane extract as a negative control. E, endogenous co-IP was performed in MERS-CoV- or mock-infected Huh7 and BEAS2B cells. Immunoprecipitation was performed using the anti-GRP78 antibody, the anti-MERS-CoV spike antibody, or the mouse isotype control. The precipitated protein complexes were detected with the anti-MERS-CoV spike antibody or the anti-GRP78 antibody. WB , Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: GRP78 interacts with the MERS-CoV spike. A, BHK21 cells were transfected with pcDNA–GRP78–V5 ( lanes 1 and 2 ) or empty vector ( lane 3 ). The cell lysate was immunoprecipitated ( IP ) with either purified recombinant MERS-CoV–S1–FLAG protein ( lanes 1 and 3 ) or E. coli bacterial alkaline phosphatase ( BAP )-FLAG protein ( lane 2 ) pre-adsorbed onto anti-FLAG M2-agarose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-V5 antibody. B, reciprocal co-IP was performed using GRP78 as the bait protein. Purified MERS-CoV–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5 Sepharose beads. The precipitated protein complex was detected using the anti-FLAG antibody or the anti-GRP78 antibody. C, membrane fraction of Huh7 cells was extracted and immunoprecipitated with either MERS-CoV–S1–FLAG( lanes 1 and 3 ) or BAP–FLAG ( lane 2 ). D, reciprocal co-IP was performed using GRP78 as the bait. Mouse IgG was used in place of the membrane extract as a negative control. E, endogenous co-IP was performed in MERS-CoV- or mock-infected Huh7 and BEAS2B cells. Immunoprecipitation was performed using the anti-GRP78 antibody, the anti-MERS-CoV spike antibody, or the mouse isotype control. The precipitated protein complexes were detected with the anti-MERS-CoV spike antibody or the anti-GRP78 antibody. WB , Western blotting.

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Recombinant, Co-Immunoprecipitation Assay, Negative Control, Infection, Western Blot

    GRP78 is up-regulated on the surface of MERS-CoV–infected cells. A, Huh7 cells were infected with MERS-CoV at 0.01 and 0.1 m.o.i. and were harvested for flow cytometry analysis at 24 h post-infection. B, percentage of MERS-CoV N–positive cells was quantified. C, in parallel, cell surface and total DPP4 and GRP78 among mock- or MERS-CoV–infected samples were analyzed by flow cytometry. D, percentage of DPP4-positive cells and GRP78-positive cells in mock- or MERS-CoV–infected samples were quantified. Total DPP4 and GRP78 staining was performed by first permeabilizing the cells with 0.1% Triton X-100, whereas the surface DPP4 and GRP78 staining was performed in the absence of cell permeabilization. The gate in A represented the percentage of MERS-CoV N–positive cells. The gates in C represented the percentage of DPP4- ( upper panels ) and GRP78 ( lower panels )-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: GRP78 is up-regulated on the surface of MERS-CoV–infected cells. A, Huh7 cells were infected with MERS-CoV at 0.01 and 0.1 m.o.i. and were harvested for flow cytometry analysis at 24 h post-infection. B, percentage of MERS-CoV N–positive cells was quantified. C, in parallel, cell surface and total DPP4 and GRP78 among mock- or MERS-CoV–infected samples were analyzed by flow cytometry. D, percentage of DPP4-positive cells and GRP78-positive cells in mock- or MERS-CoV–infected samples were quantified. Total DPP4 and GRP78 staining was performed by first permeabilizing the cells with 0.1% Triton X-100, whereas the surface DPP4 and GRP78 staining was performed in the absence of cell permeabilization. The gate in A represented the percentage of MERS-CoV N–positive cells. The gates in C represented the percentage of DPP4- ( upper panels ) and GRP78 ( lower panels )-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Infection, Flow Cytometry, Cytometry, Staining, Derivative Assay

    GRP78 interacts with the bCoV-HKU9 spike and serves as an attachment factor for bCoV-HKU9. A, BHK21 cells were transfected with pcDNA–GRP78–V5 ( lanes 1 and 2 ) or empty vector ( lane 3 ). Co-IP between GRP78 and bCoV-HKU9 spike was performed using GRP78 as the bait protein. Purified bCoV–HKU9–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated ( IP ) with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. B, co-IP between GRP78 and SARS-CoV spike was performed using GRP78 as the bait protein. Purified SARS-CoV–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. C, HKU9–S-pseudovirus entry assays were performed in a number of mammalian cell lines. Mock-inoculated and MERS–S-pseudovirus–inoculated cells were included as negative and positive controls, respectively. HKU9–S-pseudovirus and MERS–S-pseudovirus were added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 72 h post-inoculation. D, HKU9–S-pseudovirus attachment efficiency was evaluated in Caco2 and RLK cells. HKU9–S-pseudovirus was inoculated on Caco2 and RLK cells at 100 LP per cell for 2 h at 4 °C. After 2 h, the cells were washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. E, HKU9–S-pseudovirus entry in L929 and BHK21 cells was assessed with or without GRP78 overexpression. HKU9–S-pseudovirus was inoculated at 100 LP per cell for 1 h at 37 °C. Luciferase activity was determined at 72 h post-inoculation. F and G, antibody-blocking assay for HKU9–S-pseudovirus binding was performed in RLK cells. RLK cells were pre-incubated with the rabbit anti-GRP78 antibody and the rabbit control IgG from 0 to 5 μg/ml. After the pre-incubation, HKU9–S-pseudovirus was inoculated to the cells at 100 LP per cell for 2 h at 4 °C. The cells were then washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. The percentage of bCoV-HKU9 spike-positive cells was quantified in H, and the MFI of the bCoV-HKU9 spike on the cell surface was quantified in I . Gates in D, F, and G represented the percentage of HKU9 spike-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: GRP78 interacts with the bCoV-HKU9 spike and serves as an attachment factor for bCoV-HKU9. A, BHK21 cells were transfected with pcDNA–GRP78–V5 ( lanes 1 and 2 ) or empty vector ( lane 3 ). Co-IP between GRP78 and bCoV-HKU9 spike was performed using GRP78 as the bait protein. Purified bCoV–HKU9–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated ( IP ) with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. B, co-IP between GRP78 and SARS-CoV spike was performed using GRP78 as the bait protein. Purified SARS-CoV–S1–FLAG ( lanes 1 and 3 ) or BAP–FLAG proteins ( lane 2 ) were immunoprecipitated with overexpressed GRP78–V5 or pcDNA–V5 proteins pre-adsorbed on anti-V5–Sepharose beads. The precipitated protein complex was detected using the anti-V5 antibody or the anti-FLAG antibody. C, HKU9–S-pseudovirus entry assays were performed in a number of mammalian cell lines. Mock-inoculated and MERS–S-pseudovirus–inoculated cells were included as negative and positive controls, respectively. HKU9–S-pseudovirus and MERS–S-pseudovirus were added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 72 h post-inoculation. D, HKU9–S-pseudovirus attachment efficiency was evaluated in Caco2 and RLK cells. HKU9–S-pseudovirus was inoculated on Caco2 and RLK cells at 100 LP per cell for 2 h at 4 °C. After 2 h, the cells were washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. E, HKU9–S-pseudovirus entry in L929 and BHK21 cells was assessed with or without GRP78 overexpression. HKU9–S-pseudovirus was inoculated at 100 LP per cell for 1 h at 37 °C. Luciferase activity was determined at 72 h post-inoculation. F and G, antibody-blocking assay for HKU9–S-pseudovirus binding was performed in RLK cells. RLK cells were pre-incubated with the rabbit anti-GRP78 antibody and the rabbit control IgG from 0 to 5 μg/ml. After the pre-incubation, HKU9–S-pseudovirus was inoculated to the cells at 100 LP per cell for 2 h at 4 °C. The cells were then washed, fixed, and immunolabeled for flow cytometry. HKU9–S-pseudovirus binding was identified with an in-house mouse bCoV-HKU9 spike immune serum. The percentage of bCoV-HKU9 spike-positive cells was quantified in H, and the MFI of the bCoV-HKU9 spike on the cell surface was quantified in I . Gates in D, F, and G represented the percentage of HKU9 spike-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p

    Article Snippet: Rabbit anti-GRP78 from Novus Biologicals (NBP1-54318) and goat anti-CD26 from R & D Systems (AF1180) were used for antibody-blocking experiments.

    Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Purification, Immunoprecipitation, Luciferase, Activity Assay, Immunolabeling, Flow Cytometry, Cytometry, Binding Assay, Over Expression, Antibody Blocking Assay, Incubation, Derivative Assay

    SMER28 induced cocompartmentalization of APP-CTF and LC3-II. A ) N2a-APP cells were treated for 6 h with SMER28 (50 μM), and whole-cell lysates were fractionated by sucrose gradient. An aliquot of each of the 12 fractions recovered was analyzed by SDS-PAGE and Western blotting for APP-CTF, LC3, γ-adaptin, and Bip. B ) N2a-APP cells were transfected with an LC3-EGFP-containing plasmid; at 16 h post-transfection, cells were treated for 6 h with SMER28 (50 μM). Cells were fixed and immunostained with RU-369, an APP C-terminal antibody, or APLP1 antibody and imaged by confocal microscopy.

    Journal: The FASEB Journal

    Article Title: A small-molecule enhancer of autophagy decreases levels of A? and APP-CTF via Atg5-dependent autophagy pathway

    doi: 10.1096/fj.10-175158

    Figure Lengend Snippet: SMER28 induced cocompartmentalization of APP-CTF and LC3-II. A ) N2a-APP cells were treated for 6 h with SMER28 (50 μM), and whole-cell lysates were fractionated by sucrose gradient. An aliquot of each of the 12 fractions recovered was analyzed by SDS-PAGE and Western blotting for APP-CTF, LC3, γ-adaptin, and Bip. B ) N2a-APP cells were transfected with an LC3-EGFP-containing plasmid; at 16 h post-transfection, cells were treated for 6 h with SMER28 (50 μM). Cells were fixed and immunostained with RU-369, an APP C-terminal antibody, or APLP1 antibody and imaged by confocal microscopy.

    Article Snippet: The following antibodies were used at 1:1,000 dilutions: RU-369, a rabbit polyclonal antibody that recognizes the C-terminal of APP695 ( ); Ab14 antiserum targeting residues 1–25 of presenilin 1 (PS1)-NTF ( ); 6E10 antibody against Aβ1–16 (Convance, Princeton, NJ, USA); anti-LC3 (Sigma, St. Louis, MO, USA); anti-APLP1 (Calbiochem, Gibbstown, NJ, USA); anti-Beclin1 (BD Biosciences, San Jose, CA, USA); anti-PS1-CTF (Millipore, Billerica, MA, USA); anti-γ-Adaptin (BD Biosciences); and anti-Bip (Abcam, Cambridge, MA, USA).

    Techniques: SDS Page, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy

    Melatonin suppressed KA-induced ER stress in vivo. (A) Expression of GRP78, CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (B ) Relative analysis of the expression levels of GRP78, CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (C) Relative activity of calpain in KA and/or melatonin-treated animals hippocampus. ∗∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Melatonin Mediates Protective Effects against Kainic Acid-Induced Neuronal Death through Safeguarding ER Stress and Mitochondrial Disturbance

    doi: 10.3389/fnmol.2017.00049

    Figure Lengend Snippet: Melatonin suppressed KA-induced ER stress in vivo. (A) Expression of GRP78, CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (B ) Relative analysis of the expression levels of GRP78, CHOP and calpain in KA and/or melatonin-treated animals hippocampus. (C) Relative activity of calpain in KA and/or melatonin-treated animals hippocampus. ∗∗ P

    Article Snippet: The following primary antibodies were used: anti-GAPDH (1:3000, Abcam, Santa Cruz, CA, USA); anti-GRP78 (1:2000, Abcam, Cambridge, MA, USA); anti-CHOP (1:500, Abcam, Cambridge, MA, USA); anti-Mfn-1 (1:1000, Abcam, Cambridge, MA, USA), anti-Mfn-2 (1:1000, Abcam, Cambridge, MA, USA), anti-OPA-1 (1:1000, CST, Danvers, MA, USA), anti-Drp-1 (1:1000, Santa Cruz, CA, USA), anti-calpain (1:1000, Santa Cruz, CA, USA); anti-Cyt C (1:1000, Santa Cruz, CA, USA); anti-cleaved caspase-12 (1:1000, CST, Danvers, MA, USA) and anti-cleaved caspase-3 (1:1000, CST, Danvers, MA, USA), anti-cleaved caspase-9 (1:1000, CST, Danvers, MA, USA) and anti-VDAC-1(1:1500, Abcam, Cambridge, MA, USA).

    Techniques: In Vivo, Expressing, Activity Assay

    Kainic acid-induced ER stress contributes to mitochondrial defects and apoptosis and melatonin can effectively suppress ER stress. (A,B) Expression levels of GRP78, CHOP, calpain, c-caspase-12 in KA and/or melatonin treated N2a cells. (C,D) Expression levels of GRP78, CHOP, calpain, c-caspase-12 in KA and/or PBA treated N2a cells. (E,F ) Fura-2 AM probe was used to measure real-time Ca 2+ concentration in KA and/or PBA treated N2a cells. (G,H) Expression levels of Mfn-2 and c-caspase-3 in KA, KA+PBA treated N2a cells. (I) Mitochondria number in KA, KA+PBA treated N2a cells. (J) Average length of mitochondria in KA, KA+PBA treated N2a cells. (K) MMP in KA, KA+PBA treated N2a cells. (L) Relative production of ROS in KA, KA+PBA treated N2a cells. (M) Relative content of ATP in KA, KA+PBA treated N2a cells ( ∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Melatonin Mediates Protective Effects against Kainic Acid-Induced Neuronal Death through Safeguarding ER Stress and Mitochondrial Disturbance

    doi: 10.3389/fnmol.2017.00049

    Figure Lengend Snippet: Kainic acid-induced ER stress contributes to mitochondrial defects and apoptosis and melatonin can effectively suppress ER stress. (A,B) Expression levels of GRP78, CHOP, calpain, c-caspase-12 in KA and/or melatonin treated N2a cells. (C,D) Expression levels of GRP78, CHOP, calpain, c-caspase-12 in KA and/or PBA treated N2a cells. (E,F ) Fura-2 AM probe was used to measure real-time Ca 2+ concentration in KA and/or PBA treated N2a cells. (G,H) Expression levels of Mfn-2 and c-caspase-3 in KA, KA+PBA treated N2a cells. (I) Mitochondria number in KA, KA+PBA treated N2a cells. (J) Average length of mitochondria in KA, KA+PBA treated N2a cells. (K) MMP in KA, KA+PBA treated N2a cells. (L) Relative production of ROS in KA, KA+PBA treated N2a cells. (M) Relative content of ATP in KA, KA+PBA treated N2a cells ( ∗ P

    Article Snippet: The following primary antibodies were used: anti-GAPDH (1:3000, Abcam, Santa Cruz, CA, USA); anti-GRP78 (1:2000, Abcam, Cambridge, MA, USA); anti-CHOP (1:500, Abcam, Cambridge, MA, USA); anti-Mfn-1 (1:1000, Abcam, Cambridge, MA, USA), anti-Mfn-2 (1:1000, Abcam, Cambridge, MA, USA), anti-OPA-1 (1:1000, CST, Danvers, MA, USA), anti-Drp-1 (1:1000, Santa Cruz, CA, USA), anti-calpain (1:1000, Santa Cruz, CA, USA); anti-Cyt C (1:1000, Santa Cruz, CA, USA); anti-cleaved caspase-12 (1:1000, CST, Danvers, MA, USA) and anti-cleaved caspase-3 (1:1000, CST, Danvers, MA, USA), anti-cleaved caspase-9 (1:1000, CST, Danvers, MA, USA) and anti-VDAC-1(1:1500, Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Concentration Assay

    AUDA inhibited LPS-induced hyperpermeability by regulating GRP78 mediated SRC activation ( A ) LPS induced GRP78/Src interaction and Src activation was restrained by AUDA treatment; ( B ) LPS induced permeability increase was reduced by Src inhibitor PP1; ( C ) PP1 decreased LPS induced phosphorylation of VE-cadherin and MLC. ( D ) PP1 attenuated LPS induced permeability increase. ( E – G ) LPS induced VE cadherin and MLC phosphorylation was suppressed by PP1 treatment. Each tests was repeated for at least three times. Data are expressed as means ± SEM. * P

    Journal: Oncotarget

    Article Title: EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway

    doi: 10.18632/oncotarget.17331

    Figure Lengend Snippet: AUDA inhibited LPS-induced hyperpermeability by regulating GRP78 mediated SRC activation ( A ) LPS induced GRP78/Src interaction and Src activation was restrained by AUDA treatment; ( B ) LPS induced permeability increase was reduced by Src inhibitor PP1; ( C ) PP1 decreased LPS induced phosphorylation of VE-cadherin and MLC. ( D ) PP1 attenuated LPS induced permeability increase. ( E – G ) LPS induced VE cadherin and MLC phosphorylation was suppressed by PP1 treatment. Each tests was repeated for at least three times. Data are expressed as means ± SEM. * P

    Article Snippet: Antibodies including VE-cadherin Tyr-658, VE-cadherin Tyr-685 site, T-MLC, phosphorylated-MLC, T-mypt, phosphorylated-mypt and GRP78 antibody were from Abcam.

    Techniques: Activation Assay, Permeability

    Proposed model about the mechanisms by which CYP2J2 regulated LPS-induced barrier dysfunction AS indicated, LPS promoted GRP78/Src interaction and subsequent activation, leading to increased ROS generation and Rho activation, however, by suppressing GRP78/Src interaction, the CYP2J2-EETs inhibited ROS production and Rho-ROCK activation. At the same time, VE-cadherin and MLC phosphorylation was also interrupted, thus suppressed LPS induced barrier dysfunction eventually.

    Journal: Oncotarget

    Article Title: EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway

    doi: 10.18632/oncotarget.17331

    Figure Lengend Snippet: Proposed model about the mechanisms by which CYP2J2 regulated LPS-induced barrier dysfunction AS indicated, LPS promoted GRP78/Src interaction and subsequent activation, leading to increased ROS generation and Rho activation, however, by suppressing GRP78/Src interaction, the CYP2J2-EETs inhibited ROS production and Rho-ROCK activation. At the same time, VE-cadherin and MLC phosphorylation was also interrupted, thus suppressed LPS induced barrier dysfunction eventually.

    Article Snippet: Antibodies including VE-cadherin Tyr-658, VE-cadherin Tyr-685 site, T-MLC, phosphorylated-MLC, T-mypt, phosphorylated-mypt and GRP78 antibody were from Abcam.

    Techniques: Activation Assay

    Knocking-down the expression of GRP78 does not affect the replication of JE viral RNA . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (scramble siRNA) for 48 hours. The expression level of GRP78 was measured by Western blot analysis using polyclonal antibody specific to GRP78. Transfected cells were then infected with JEV at an MOI of 1. At 24 hour post-infection, the cell lysates were collected to measure the JEV replication using antibodies specific to NS1 and/or NS5. Two independent replicates are shown for the scramble and GRP78 siRNA conditions

    Journal: Virology Journal

    Article Title: Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

    doi: 10.1186/1743-422X-8-128

    Figure Lengend Snippet: Knocking-down the expression of GRP78 does not affect the replication of JE viral RNA . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (scramble siRNA) for 48 hours. The expression level of GRP78 was measured by Western blot analysis using polyclonal antibody specific to GRP78. Transfected cells were then infected with JEV at an MOI of 1. At 24 hour post-infection, the cell lysates were collected to measure the JEV replication using antibodies specific to NS1 and/or NS5. Two independent replicates are shown for the scramble and GRP78 siRNA conditions

    Article Snippet: The mixture was then added with 4 mL cell growth medium for 2 days, and the GRP78 protein was detected in siRNA transfected cells by Western blot using anti-GRP78 specific antibody (Abcam, Cambridge, MA).

    Techniques: Expressing, Transfection, Western Blot, Infection

    Decrease in JEV infectivity in the absence of co-migrating GRP78 . A) Viral infectivity using plaque assay of JE virion-fractions associated without or with GRP78 was determined. B) Quantitative measurement of viral progeny produced from E+GRP78 and E only fractionates. The plaque assay results shown here are representatives of three independent experiments.

    Journal: Virology Journal

    Article Title: Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

    doi: 10.1186/1743-422X-8-128

    Figure Lengend Snippet: Decrease in JEV infectivity in the absence of co-migrating GRP78 . A) Viral infectivity using plaque assay of JE virion-fractions associated without or with GRP78 was determined. B) Quantitative measurement of viral progeny produced from E+GRP78 and E only fractionates. The plaque assay results shown here are representatives of three independent experiments.

    Article Snippet: The mixture was then added with 4 mL cell growth medium for 2 days, and the GRP78 protein was detected in siRNA transfected cells by Western blot using anti-GRP78 specific antibody (Abcam, Cambridge, MA).

    Techniques: Infection, Plaque Assay, Produced

    GRP78 is released into the media upon JEV infection and partially co-fractionates with the JE virion . A) Verification of GRP78 in the secretome from JEV-induced BHK-21 cells. Cell lysates or secretion medium were collected 3 days post-infection followed by SDS-PAGE for protein separation. The GRP78 was detected by anti-GRP78 specific antibody. Two independent replicates are shown for the mock- and JEV-infected conditions. B) Sucrose density gradient fraction of JE virion and GPR78. A volume of 40 μL of sample from each fraction was analyzed on SDS-PAGE followed by the detection of anti-JEV E protein and anti-GRP78 by Western blotting.

    Journal: Virology Journal

    Article Title: Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

    doi: 10.1186/1743-422X-8-128

    Figure Lengend Snippet: GRP78 is released into the media upon JEV infection and partially co-fractionates with the JE virion . A) Verification of GRP78 in the secretome from JEV-induced BHK-21 cells. Cell lysates or secretion medium were collected 3 days post-infection followed by SDS-PAGE for protein separation. The GRP78 was detected by anti-GRP78 specific antibody. Two independent replicates are shown for the mock- and JEV-infected conditions. B) Sucrose density gradient fraction of JE virion and GPR78. A volume of 40 μL of sample from each fraction was analyzed on SDS-PAGE followed by the detection of anti-JEV E protein and anti-GRP78 by Western blotting.

    Article Snippet: The mixture was then added with 4 mL cell growth medium for 2 days, and the GRP78 protein was detected in siRNA transfected cells by Western blot using anti-GRP78 specific antibody (Abcam, Cambridge, MA).

    Techniques: Infection, SDS Page, Western Blot

    The co-localization of GRP78 and JEV E protein in JEV-infected BHK-21 cells . Mock- or JEV-infected BHK-21 cells were harvested at 3 days post-infection and prepared for immunofluorescence analysis stained with antibodies that detect GRP78 (green; b, f, j) and JEV-E protein (red; a, e, i).

    Journal: Virology Journal

    Article Title: Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

    doi: 10.1186/1743-422X-8-128

    Figure Lengend Snippet: The co-localization of GRP78 and JEV E protein in JEV-infected BHK-21 cells . Mock- or JEV-infected BHK-21 cells were harvested at 3 days post-infection and prepared for immunofluorescence analysis stained with antibodies that detect GRP78 (green; b, f, j) and JEV-E protein (red; a, e, i).

    Article Snippet: The mixture was then added with 4 mL cell growth medium for 2 days, and the GRP78 protein was detected in siRNA transfected cells by Western blot using anti-GRP78 specific antibody (Abcam, Cambridge, MA).

    Techniques: Infection, Immunofluorescence, Staining

    Knocking-down the expression of GRP78 by siRNA decreases the yield of infectious JE virus production . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (negative control). The down-regulation of GRP78 was measured by Western blot analysis using antibody specific to GRP78 as shown in Figure 5. A) Transfected cell lysates were then infected with JEV at an MOI of 1. At 24 hours post-infection, the supernatants were collected to measure the amount of JE viral RNA production by RT real-time PCR as described in Material and Methods. The virus yield is expressed as a percentage of the yield obtained from cells transfected with irrelevant siRNA. B) Plaque formation by JE virus-particle collected from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. C) Quantitative measurement of viral progeny produced from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. The virus titer is defined as plaque-forming unit (PFU) per mL. Results are derived from three independent experiments.

    Journal: Virology Journal

    Article Title: Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity

    doi: 10.1186/1743-422X-8-128

    Figure Lengend Snippet: Knocking-down the expression of GRP78 by siRNA decreases the yield of infectious JE virus production . BHK-21 cells were transfected with siRNA against GRP78 or an irrelevant siRNA (negative control). The down-regulation of GRP78 was measured by Western blot analysis using antibody specific to GRP78 as shown in Figure 5. A) Transfected cell lysates were then infected with JEV at an MOI of 1. At 24 hours post-infection, the supernatants were collected to measure the amount of JE viral RNA production by RT real-time PCR as described in Material and Methods. The virus yield is expressed as a percentage of the yield obtained from cells transfected with irrelevant siRNA. B) Plaque formation by JE virus-particle collected from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. C) Quantitative measurement of viral progeny produced from JEV-infected scramble siRNA treated cells or cells treated with siRNA against GRP78. The virus titer is defined as plaque-forming unit (PFU) per mL. Results are derived from three independent experiments.

    Article Snippet: The mixture was then added with 4 mL cell growth medium for 2 days, and the GRP78 protein was detected in siRNA transfected cells by Western blot using anti-GRP78 specific antibody (Abcam, Cambridge, MA).

    Techniques: Expressing, Transfection, Negative Control, Western Blot, Infection, Real-time Polymerase Chain Reaction, Produced, Derivative Assay

    Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p

    Article Snippet: Huh7 and RLK cells grown in 96-well plates were washed twice with PBS (ThermoFisher Scientific) and incubated with neuraminidase from Clostridium perfringens (Sigma) diluted in FBS-free growth medium at 37 °C for 3 h. After the incubation, the cells were washed three times and challenged with MERS–S- or HKU9–S-pseudoviruses, with or without pre-incubation with the GRP78 polyclonal antibody (Abcam) for 1 h at 37 °C.

    Techniques: Activated Clotting Time Assay, Incubation, Infection, Standard Deviation, Derivative Assay

    Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus ( A ), bronchiole ( B ), and alveolus ( C ). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D . Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D .

    Journal: The Journal of Biological Chemistry

    Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

    doi: 10.1074/jbc.RA118.001897

    Figure Lengend Snippet: Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus ( A ), bronchiole ( B ), and alveolus ( C ). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D . Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D .

    Article Snippet: Huh7 and RLK cells grown in 96-well plates were washed twice with PBS (ThermoFisher Scientific) and incubated with neuraminidase from Clostridium perfringens (Sigma) diluted in FBS-free growth medium at 37 °C for 3 h. After the incubation, the cells were washed three times and challenged with MERS–S- or HKU9–S-pseudoviruses, with or without pre-incubation with the GRP78 polyclonal antibody (Abcam) for 1 h at 37 °C.

    Techniques: Expressing, Immunostaining, Labeling

    Alteration of the expressions of apoptosis-associated mitochondrial and ER stress proteins in the bladder among different experimental groups. ( A – E ) The apoptosis of bladder cells as detected by TUNEL, (FITC, green) and DAPI staining (blue) for UL and SL. There were increases in TUNEL - positive nuclei (white arrows) in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. Scale bar = 100 μm. ( F – I ) The expression levels of pro-apoptotic and anti-apoptotic proteins in the bladder tissue by western blots. ( F , G ) Quantifications of the ER stress protein expressions to β-actin. The expression levels of GRP78, CHOP and caspase-12 slightly were increased in the MetS group, and significantly promoted in the MetS + OVX and MetS + OVX + EGCG groups. ( H , I ) Quantifications of mitochondrial protein expressions to β-actin. The expression of Bcl-2 was decreased in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. In contrast, the pro-apoptosis expressions were meaningfully increased in those groups. Results were normalized as the control = 100%. Values represented the mean ± SD for n = 8. * P

    Journal: Scientific Reports

    Article Title: Epigallocatechin-3-gallate alleviates bladder overactivity in a rat model with metabolic syndrome and ovarian hormone deficiency through mitochondria apoptosis pathways

    doi: 10.1038/s41598-018-23800-w

    Figure Lengend Snippet: Alteration of the expressions of apoptosis-associated mitochondrial and ER stress proteins in the bladder among different experimental groups. ( A – E ) The apoptosis of bladder cells as detected by TUNEL, (FITC, green) and DAPI staining (blue) for UL and SL. There were increases in TUNEL - positive nuclei (white arrows) in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. Scale bar = 100 μm. ( F – I ) The expression levels of pro-apoptotic and anti-apoptotic proteins in the bladder tissue by western blots. ( F , G ) Quantifications of the ER stress protein expressions to β-actin. The expression levels of GRP78, CHOP and caspase-12 slightly were increased in the MetS group, and significantly promoted in the MetS + OVX and MetS + OVX + EGCG groups. ( H , I ) Quantifications of mitochondrial protein expressions to β-actin. The expression of Bcl-2 was decreased in the MetS, MetS + OVX, and MetS + OVX + EGCG groups. In contrast, the pro-apoptosis expressions were meaningfully increased in those groups. Results were normalized as the control = 100%. Values represented the mean ± SD for n = 8. * P

    Article Snippet: Western blot results showed that the expression levels of CHOP, GRP78 and Caspase-12 were significantly increased by 1.3-fold, 2.2-fold and 1.3-fold of control, respectively, when the MetS group was compared with the control group.

    Techniques: TUNEL Assay, Staining, Expressing, Western Blot

    Proposed mechanistic model for MetS and ovarian hormone deficiency - induced oxidative stress through mitochondria and ER - mediated apoptosis pathways and the potential effect of EGCG on bladder overactivity. MetS and ovarian hormone deficiency induced mitochondria to release cytochrome C and caspase activation (caspase 9 and 3), and promoted ER to release GRP78, CHOP and caspase 12 to induce the generation of oxidative stress and apoptosis However, pretreatment attenuated oxidative stress induced by MetS or/and ovarian hormone deficiency, and lessened the expression of mitochondrial and ER apoptotic signals. (HFHS, high fat high sugar dieting; OVX, Ovariectomy; ER, Endoplasmic reticulum).

    Journal: Scientific Reports

    Article Title: Epigallocatechin-3-gallate alleviates bladder overactivity in a rat model with metabolic syndrome and ovarian hormone deficiency through mitochondria apoptosis pathways

    doi: 10.1038/s41598-018-23800-w

    Figure Lengend Snippet: Proposed mechanistic model for MetS and ovarian hormone deficiency - induced oxidative stress through mitochondria and ER - mediated apoptosis pathways and the potential effect of EGCG on bladder overactivity. MetS and ovarian hormone deficiency induced mitochondria to release cytochrome C and caspase activation (caspase 9 and 3), and promoted ER to release GRP78, CHOP and caspase 12 to induce the generation of oxidative stress and apoptosis However, pretreatment attenuated oxidative stress induced by MetS or/and ovarian hormone deficiency, and lessened the expression of mitochondrial and ER apoptotic signals. (HFHS, high fat high sugar dieting; OVX, Ovariectomy; ER, Endoplasmic reticulum).

    Article Snippet: Western blot results showed that the expression levels of CHOP, GRP78 and Caspase-12 were significantly increased by 1.3-fold, 2.2-fold and 1.3-fold of control, respectively, when the MetS group was compared with the control group.

    Techniques: Activation Assay, Expressing

    The combination of PTN and three required binding partners promotes DIPG invasion toward SVZ hNPC CM . (A) No single candidate recombinant protein significantly increased DIPG invasion compared to unconditioned hNPC media. (B) The combination of four factors: PTN, SPARC, SPARCL1, and HSP90B, was sufficient for the full invasion-promoting effect toward SVZ hNPC CM. No combination of two or three factors was sufficient. All experiments performed with n = 3 replicates/wells in SU-DIPG-XIII FL cells and analyzed by one-way ANOVA with Tukey post hoc adjustment for multiple comparisons (A) or Dunnett post hoc adjustment for multiple comparisons to either SVZ hNPC CM or the combination of all 4 factors (B). (C) All four proteins coeluted at approximately the 212 kDa size expected for a complex of all four proteins by size exclusion chromatography. (D) All four proteins copurified together in immunoprecipitation reactions for any one of the four proteins, more so than with a control IgG. Three control proteins also present in SVZ hNPC CM: GRP78, IGFBP2, and BCAN, did not copurify with any of the four proteins. ) invade preferentially toward the combination of four proteins similarly to toward human SVZ NPC CM, compared to unconditioned hNPC media. All experiments performed with n = 3 replicates/wells and analyzed by unpaired, two-tailed Student’s t-tests for comparison between unconditioned and conditioned hNPC media. Data shown as mean ± SEM. *p

    Journal: Cell

    Article Title: Neural precursor-derived pleiotrophin mediates subventricular zone invasion by glioma

    doi: 10.1016/j.cell.2017.07.016

    Figure Lengend Snippet: The combination of PTN and three required binding partners promotes DIPG invasion toward SVZ hNPC CM . (A) No single candidate recombinant protein significantly increased DIPG invasion compared to unconditioned hNPC media. (B) The combination of four factors: PTN, SPARC, SPARCL1, and HSP90B, was sufficient for the full invasion-promoting effect toward SVZ hNPC CM. No combination of two or three factors was sufficient. All experiments performed with n = 3 replicates/wells in SU-DIPG-XIII FL cells and analyzed by one-way ANOVA with Tukey post hoc adjustment for multiple comparisons (A) or Dunnett post hoc adjustment for multiple comparisons to either SVZ hNPC CM or the combination of all 4 factors (B). (C) All four proteins coeluted at approximately the 212 kDa size expected for a complex of all four proteins by size exclusion chromatography. (D) All four proteins copurified together in immunoprecipitation reactions for any one of the four proteins, more so than with a control IgG. Three control proteins also present in SVZ hNPC CM: GRP78, IGFBP2, and BCAN, did not copurify with any of the four proteins. ) invade preferentially toward the combination of four proteins similarly to toward human SVZ NPC CM, compared to unconditioned hNPC media. All experiments performed with n = 3 replicates/wells and analyzed by unpaired, two-tailed Student’s t-tests for comparison between unconditioned and conditioned hNPC media. Data shown as mean ± SEM. *p

    Article Snippet: Primary antibodies used were: mouse anti-pleiotrophin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-SPARC (1:100; R & D Systems, Minneapolis, MN), goat anti-SPARCL1 (1:000; R & D Systems, Minneapolis, MN), rabbit anti-HSP90B (GeneTex, Irvine, CA), rabbit anti-GRP78 (1:500, Abcam, Cambridge, MA), rabbit anti-PTPRZ (Thermo Fisher, Waltham, MA), and rabbit beta-actin (1:2000; Cell Signaling, Danvers, MA).

    Techniques: Binding Assay, Recombinant, Size-exclusion Chromatography, Immunoprecipitation, Two Tailed Test

    Expression patterns of unfolded protein response ( UPR ) signal genes in cumulus‐oocyte complexes ( COC s), denuded oocyte ( DO ), and cumulus cells ( CC ) on porcine oocytes in vitro maturation ( IVM ) (22 and 44 h). A, The mRNA levels of activated UPR signal transcription factors ( Bip/Grp78 and Atf4 ) on maturing COC s, DO , and CC of porcine IVM process (metaphase, M I; 22 h and metaphase, M II ; 44 h) were measured by reverse transcription‐polymerase chain reaction ( PCR ) ( RT ‐ PCR ) analysis. Relative folds of Bip/Grp78 and Atf4 were obtained by normalizing the signals for Gapdh . B‐C, Western blotting results of Bip/Grp78, ATF 4, P50 ATF 6, and CHOP in DO , COC s, and CC were compared at the M I (22 h) and M II (44 h) stages of pig oocyte maturation. Relative folds of UPR marker protein levels were obtained by normalizing the signals for β‐Actin. D, Immunohistochemistry ( IHC ) staining of P50 ATF 6 in pig ovaries with different follicle sizes (1‐2 mm, small; 3‐4 mm, middle; and 5‐6 mm, large). IHC staining for P50 ATF 6 is detected using specific P50 ATF 6 antibody in in vivo ‐ maturing oocytes of pig ovary follicles. O = immature oocyte, Scale bar = 200 μm. Histograms represent values of densitometry analysis obtained using ImageJ software. Data in the bar graph are means ± SEM / SD of three independent experiments (per 50 DO s and 30 COC s). *** P

    Journal: Journal of Pineal Research

    Article Title: Melatonin improves the meiotic maturation of porcine oocytes by reducing endoplasmic reticulum stress during in vitro maturation, et al. Melatonin improves the meiotic maturation of porcine oocytes by reducing endoplasmic reticulum stress during in vitro maturation

    doi: 10.1111/jpi.12458

    Figure Lengend Snippet: Expression patterns of unfolded protein response ( UPR ) signal genes in cumulus‐oocyte complexes ( COC s), denuded oocyte ( DO ), and cumulus cells ( CC ) on porcine oocytes in vitro maturation ( IVM ) (22 and 44 h). A, The mRNA levels of activated UPR signal transcription factors ( Bip/Grp78 and Atf4 ) on maturing COC s, DO , and CC of porcine IVM process (metaphase, M I; 22 h and metaphase, M II ; 44 h) were measured by reverse transcription‐polymerase chain reaction ( PCR ) ( RT ‐ PCR ) analysis. Relative folds of Bip/Grp78 and Atf4 were obtained by normalizing the signals for Gapdh . B‐C, Western blotting results of Bip/Grp78, ATF 4, P50 ATF 6, and CHOP in DO , COC s, and CC were compared at the M I (22 h) and M II (44 h) stages of pig oocyte maturation. Relative folds of UPR marker protein levels were obtained by normalizing the signals for β‐Actin. D, Immunohistochemistry ( IHC ) staining of P50 ATF 6 in pig ovaries with different follicle sizes (1‐2 mm, small; 3‐4 mm, middle; and 5‐6 mm, large). IHC staining for P50 ATF 6 is detected using specific P50 ATF 6 antibody in in vivo ‐ maturing oocytes of pig ovary follicles. O = immature oocyte, Scale bar = 200 μm. Histograms represent values of densitometry analysis obtained using ImageJ software. Data in the bar graph are means ± SEM / SD of three independent experiments (per 50 DO s and 30 COC s). *** P

    Article Snippet: After blocking, the membranes were incubated with anti‐Bip/Grp78 (1:2000, Catalog number SC‐1050; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐ATF4 (1:5000, Catalog number SC‐200; Santa Cruz), anti‐P90/50 ATF6 (1:4000, Catalog number NBP1‐40256; Novus Biologicals, Littleton, CO, USA), anti‐CHOP (1:500, Catalog number SC‐793; Santa Cruz), and anti‐β‐Actin (1:3000, Catalog number SC‐47778; Santa Cruz) antibodies.

    Techniques: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Marker, Immunohistochemistry, Staining, In Vivo, Software

    Cab45S increases the GRP78/BiP protein level. ( a and d ) Western blots of GRP78/BiP and two other ER molecular chaperones, PDI and calnexin, in stable Cab45S-knockdown ( a ) or Cab45S-overexpressed ( d ) PANC-1 cell lines. Numbers represent different cell lines. ( b and c ) Western blots ( b ) and quantification ( c ) of GRP78/BiP in Cab45S-knockdown and control (shNC, scrambled shRNA) HeLa cells treated with TM (2 μ g/ml) for the indicated periods. GAPDH was used as a loading control. ( e ) Quantitative real-time PCR of the relative GRP78/BiP mRNA expression levels in Cab45S-knockdown and control HeLa cells treated with TM for the indicated times ( n =3). ( f ) Western blots of GRP78/BiP in Cab45S-knockdown and control HeLa cells treated with TM (2 μ g/ml, 4 h) followed by cycloheximide (Chx; 100 μ M) for the indicated periods. For c and e , data are presented as mean±S.E.M. ** P

    Journal: Cell Death & Disease

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    doi: 10.1038/cddis.2014.193

    Figure Lengend Snippet: Cab45S increases the GRP78/BiP protein level. ( a and d ) Western blots of GRP78/BiP and two other ER molecular chaperones, PDI and calnexin, in stable Cab45S-knockdown ( a ) or Cab45S-overexpressed ( d ) PANC-1 cell lines. Numbers represent different cell lines. ( b and c ) Western blots ( b ) and quantification ( c ) of GRP78/BiP in Cab45S-knockdown and control (shNC, scrambled shRNA) HeLa cells treated with TM (2 μ g/ml) for the indicated periods. GAPDH was used as a loading control. ( e ) Quantitative real-time PCR of the relative GRP78/BiP mRNA expression levels in Cab45S-knockdown and control HeLa cells treated with TM for the indicated times ( n =3). ( f ) Western blots of GRP78/BiP in Cab45S-knockdown and control HeLa cells treated with TM (2 μ g/ml, 4 h) followed by cycloheximide (Chx; 100 μ M) for the indicated periods. For c and e , data are presented as mean±S.E.M. ** P

    Article Snippet: Regents and antibodies The primary antibodies were anti-BrdU (MBL, Nagoya, Japan), anti-GFP (MBL), anti-Flag (Sigma), anti-tubulin (Sigma), anti-ATF4 (GeneTex, San Antonio, TX, USA) and anti-PARP, anti-calnexin, anti-PDI, anti-p-JNK, anti-caspase-3, anti-PERK, anti-IRE1, anti-GAPDH, anti-pIRE1 and anti-GRP78/BiP from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Western Blot, shRNA, Real-time Polymerase Chain Reaction, Expressing

    Cab45S inhibits ER stress-induced apoptosis via GRP78/BiP. ( a ) MTS assay of viable HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP treated with TM (2 μ g/ml, 48 h; n =3). ( b ) MTS assay of viable HeLa cells transfected with vectors expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP treated with TM (2 μ g/ml, 48 h; n =3). ( c ) Representative photomicrographs from TUNEL assay of apoptotic HeLa cells transfected with vectors expressing 3 × Flag and shNC, 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP treated with TM (2 μ g/ml, 48 h). Scale bar, 100 μ m. ( d ) Quantification of TUNEL-positive cells as in c ( n =3; > 100 cells per experiment). ( e ) Representative photomicrographs from TUNEL assay of apoptotic HeLa cells transfected with vectors expressing shNC and EGFP, shCab45S and EGFP, or shCab45S and GRP78/BiP-EGFP treated with TM (2 μ g/ml, 48 h). Scale bar, 100 μ m. ( f ) Quantification of TUNEL-positive cells as in e ( n =3; > 100 cells per experiment). For a , b , d and f , data are presented as mean±S.E.M. ** P

    Journal: Cell Death & Disease

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    doi: 10.1038/cddis.2014.193

    Figure Lengend Snippet: Cab45S inhibits ER stress-induced apoptosis via GRP78/BiP. ( a ) MTS assay of viable HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP treated with TM (2 μ g/ml, 48 h; n =3). ( b ) MTS assay of viable HeLa cells transfected with vectors expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP treated with TM (2 μ g/ml, 48 h; n =3). ( c ) Representative photomicrographs from TUNEL assay of apoptotic HeLa cells transfected with vectors expressing 3 × Flag and shNC, 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP treated with TM (2 μ g/ml, 48 h). Scale bar, 100 μ m. ( d ) Quantification of TUNEL-positive cells as in c ( n =3; > 100 cells per experiment). ( e ) Representative photomicrographs from TUNEL assay of apoptotic HeLa cells transfected with vectors expressing shNC and EGFP, shCab45S and EGFP, or shCab45S and GRP78/BiP-EGFP treated with TM (2 μ g/ml, 48 h). Scale bar, 100 μ m. ( f ) Quantification of TUNEL-positive cells as in e ( n =3; > 100 cells per experiment). For a , b , d and f , data are presented as mean±S.E.M. ** P

    Article Snippet: Regents and antibodies The primary antibodies were anti-BrdU (MBL, Nagoya, Japan), anti-GFP (MBL), anti-Flag (Sigma), anti-tubulin (Sigma), anti-ATF4 (GeneTex, San Antonio, TX, USA) and anti-PARP, anti-calnexin, anti-PDI, anti-p-JNK, anti-caspase-3, anti-PERK, anti-IRE1, anti-GAPDH, anti-pIRE1 and anti-GRP78/BiP from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: MTS Assay, Transfection, Expressing, shRNA, TUNEL Assay

    Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

    Journal: Cell Death & Disease

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    doi: 10.1038/cddis.2014.193

    Figure Lengend Snippet: Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

    Article Snippet: Regents and antibodies The primary antibodies were anti-BrdU (MBL, Nagoya, Japan), anti-GFP (MBL), anti-Flag (Sigma), anti-tubulin (Sigma), anti-ATF4 (GeneTex, San Antonio, TX, USA) and anti-PARP, anti-calnexin, anti-PDI, anti-p-JNK, anti-caspase-3, anti-PERK, anti-IRE1, anti-GAPDH, anti-pIRE1 and anti-GRP78/BiP from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Binding Assay, Immunoprecipitation, Expressing, SDS Page, Mass Spectrometry, Transfection

    Cab45S inhibits IRE1 activation via GRP78/BiP. ( a and b ) Western blots ( a ) and quantification ( b ) of p-IRE1 in HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( c and d ) Western blots ( c ) and quantification ( d ) of p-IRE1 in HeLa cells expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( e ) Extracts of HEK293T cells overexpressing the indicated vectors treated with TM (1 μ g/ml, 24 h) were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag, anti-EGFP or anti-Cab45S antibody. ( f ) Working model of the mechanism by which Cab45S controls ER stress-induced apoptosis. Cab45S interacts with the NBD of GRP78/BiP, which prevents its disassociation from IRE1 and increases the protein level of GRP78/BiP. These effects lead to inhibition of the IRE1-JNK pathway and ER stress-induced apoptosis. For b and d , data are presented as mean±S.E.M. ** P

    Journal: Cell Death & Disease

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    doi: 10.1038/cddis.2014.193

    Figure Lengend Snippet: Cab45S inhibits IRE1 activation via GRP78/BiP. ( a and b ) Western blots ( a ) and quantification ( b ) of p-IRE1 in HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( c and d ) Western blots ( c ) and quantification ( d ) of p-IRE1 in HeLa cells expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( e ) Extracts of HEK293T cells overexpressing the indicated vectors treated with TM (1 μ g/ml, 24 h) were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag, anti-EGFP or anti-Cab45S antibody. ( f ) Working model of the mechanism by which Cab45S controls ER stress-induced apoptosis. Cab45S interacts with the NBD of GRP78/BiP, which prevents its disassociation from IRE1 and increases the protein level of GRP78/BiP. These effects lead to inhibition of the IRE1-JNK pathway and ER stress-induced apoptosis. For b and d , data are presented as mean±S.E.M. ** P

    Article Snippet: Regents and antibodies The primary antibodies were anti-BrdU (MBL, Nagoya, Japan), anti-GFP (MBL), anti-Flag (Sigma), anti-tubulin (Sigma), anti-ATF4 (GeneTex, San Antonio, TX, USA) and anti-PARP, anti-calnexin, anti-PDI, anti-p-JNK, anti-caspase-3, anti-PERK, anti-IRE1, anti-GAPDH, anti-pIRE1 and anti-GRP78/BiP from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Activation Assay, Western Blot, Transfection, Expressing, shRNA, Immunoprecipitation, Inhibition