Antibody data
- Antibody Data
- Antigen structure
- References [29]
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- Validations
- Western blot [2]
- Immunocytochemistry [6]
- Other assay [14]
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- Product number
- PA1-014A - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- GRP78 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- PA1-014A detects glucose regulated protein/BiP (GRP78) human, rat, mouse, and canine samples. PA1-014A has been successfully used in Western blot and immunofluorescence procedures. By Western blot, this antibody detects a 78 kDa protein representing GRP78 from rat liver lysates. The PA1-014A immunogen is a synthetic peptide corresponding to residues C T(643) G E E D T S E K D E L(654) of rat GRP78. Reconstitute with PBS.
- Reactivity
- Human, Mouse, Rat, Canine
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 1 mg/mL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
Submitted references Missense PNLIP mutations impeding pancreatic lipase secretion cause protein misfolding and endoplasmic reticulum stress.
Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.
Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.
Osteoblastic Swedish mutant APP expedites brain deficits by inducing endoplasmic reticulum stress-driven senescence.
Protective Effect of Anthocyanin-Enriched Polyphenols from Hibiscus syriacus L. (Malvaceae) against Ultraviolet B-Induced Damage.
Novel role of dynamin-related-protein 1 in dynamics of ER-lipid droplets in adipose tissue.
Omega-3 Fatty Acid-Enriched Fish Oil and Selenium Combination Modulates Endoplasmic Reticulum Stress Response Elements and Reverses Acquired Gefitinib Resistance in HCC827 Lung Adenocarcinoma Cells.
Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification.
Proteomic Analysis of Placental Mitochondria Following Trophoblast Differentiation.
A mechanical acupuncture instrument mitigates the endoplasmic reticulum stress and oxidative stress of ovariectomized rats.
GRP78 translocation to the cell surface and O-GlcNAcylation of VE-Cadherin contribute to ER stress-mediated endothelial permeability.
Opposite Regulation of CHOP and GRP78 and Synergistic Apoptosis Induction by Selenium Yeast and Fish Oil via AMPK Activation in Lung Adenocarcinoma Cells.
Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation-induced pancreatitis.
Featured Article: Deterioration of visual function mediated by senescence-associated endoplasmic reticulum stress in inflammatory tie2-TNF mice.
NCOA3 coactivator is a transcriptional target of XBP1 and regulates PERK-eIF2α-ATF4 signalling in breast cancer.
A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.
PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response.
Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC).
Proteomic analysis of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor complexes.
Ras inhibits endoplasmic reticulum stress in human cancer cells with amplified Myc.
Dysferlin overexpression in skeletal muscle produces a progressive myopathy.
HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.
Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor.
Membrane topology of the murine fatty acid transport protein 1.
Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes.
Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes.
Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.
Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.
Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.
Toldi V, Kassay N, Szabó A
Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2021 Oct;21(7):1317-1325
Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2021 Oct;21(7):1317-1325
Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.
George G, Ninagawa S, Yagi H, Furukawa JI, Hashii N, Ishii-Watabe A, Deng Y, Matsushita K, Ishikawa T, Mamahit YP, Maki Y, Kajihara Y, Kato K, Okada T, Mori K
eLife 2021 Oct 26;10
eLife 2021 Oct 26;10
Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.
Tsai SJ, Atai NA, Cacciottolo M, Nice J, Salehi A, Guo C, Sedgwick A, Kanagavelu S, Gould SJ
The Journal of biological chemistry 2021 Nov;297(5):101266
The Journal of biological chemistry 2021 Nov;297(5):101266
Osteoblastic Swedish mutant APP expedites brain deficits by inducing endoplasmic reticulum stress-driven senescence.
Pan JX, Sun D, Lee D, Xiong L, Ren X, Guo HH, Yao LL, Lu Y, Jung C, Xiong WC
Communications biology 2021 Nov 25;4(1):1326
Communications biology 2021 Nov 25;4(1):1326
Protective Effect of Anthocyanin-Enriched Polyphenols from Hibiscus syriacus L. (Malvaceae) against Ultraviolet B-Induced Damage.
Karunarathne WAHM, Molagoda IMN, Lee KT, Choi YH, Yu SM, Kang CH, Kim GY
Antioxidants (Basel, Switzerland) 2021 Apr 9;10(4)
Antioxidants (Basel, Switzerland) 2021 Apr 9;10(4)
Novel role of dynamin-related-protein 1 in dynamics of ER-lipid droplets in adipose tissue.
Li X, Yang L, Mao Z, Pan X, Zhao Y, Gu X, Eckel-Mahan K, Zuo Z, Tong Q, Hartig SM, Cheng X, Du G, Moore DD, Bellen HJ, Sesaki H, Sun K
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 Jun;34(6):8265-8282
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 Jun;34(6):8265-8282
Omega-3 Fatty Acid-Enriched Fish Oil and Selenium Combination Modulates Endoplasmic Reticulum Stress Response Elements and Reverses Acquired Gefitinib Resistance in HCC827 Lung Adenocarcinoma Cells.
Liao CH, Tzeng YT, Lai GM, Chang CL, Hu MH, Tsai WL, Liu YR, Hsia S, Chuang SE, Chiou TJ, Wang LM, Whang-Peng J, Yao CJ
Marine drugs 2020 Jul 29;18(8)
Marine drugs 2020 Jul 29;18(8)
Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification.
Santos MCFD, Anderson CP, Neschen S, Zumbrennen-Bullough KB, Romney SJ, Kahle-Stephan M, Rathkolb B, Gailus-Durner V, Fuchs H, Wolf E, Rozman J, de Angelis MH, Cai WM, Rajan M, Hu J, Dedon PC, Leibold EA
Nature communications 2020 Jan 15;11(1):296
Nature communications 2020 Jan 15;11(1):296
Proteomic Analysis of Placental Mitochondria Following Trophoblast Differentiation.
Fisher JJ, McKeating DR, Cuffe JS, Bianco-Miotto T, Holland OJ, Perkins AV
Frontiers in physiology 2019;10:1536
Frontiers in physiology 2019;10:1536
A mechanical acupuncture instrument mitigates the endoplasmic reticulum stress and oxidative stress of ovariectomized rats.
Seo SY, Kang SY, Kwon OS, Bang SK, Kim SP, Choi KH, Moon JY, Ryu Y
Integrative medicine research 2019 Sep;8(3):187-194
Integrative medicine research 2019 Sep;8(3):187-194
GRP78 translocation to the cell surface and O-GlcNAcylation of VE-Cadherin contribute to ER stress-mediated endothelial permeability.
Lenin R, Nagy PG, Jha KA, Gangaraju R
Scientific reports 2019 Jul 25;9(1):10783
Scientific reports 2019 Jul 25;9(1):10783
Opposite Regulation of CHOP and GRP78 and Synergistic Apoptosis Induction by Selenium Yeast and Fish Oil via AMPK Activation in Lung Adenocarcinoma Cells.
Kao RH, Lai GM, Chow JM, Liao CH, Zheng YM, Tsai WL, Hsia S, Lai IC, Lee HL, Chuang SE, Whang-Peng J, Yao CJ
Nutrients 2018 Oct 8;10(10)
Nutrients 2018 Oct 8;10(10)
Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation-induced pancreatitis.
Dolai S, Liang T, Orabi AI, Xie L, Holmyard D, Javed TA, Fernandez NA, Xie H, Cattral MS, Thurmond DC, Thorn P, Gaisano HY
The Journal of biological chemistry 2018 Feb 16;293(7):2510-2522
The Journal of biological chemistry 2018 Feb 16;293(7):2510-2522
Featured Article: Deterioration of visual function mediated by senescence-associated endoplasmic reticulum stress in inflammatory tie2-TNF mice.
Lenin R, Nagy PG, Gentry J, Gangaraju R
Experimental biology and medicine (Maywood, N.J.) 2018 Aug;243(12):976-984
Experimental biology and medicine (Maywood, N.J.) 2018 Aug;243(12):976-984
NCOA3 coactivator is a transcriptional target of XBP1 and regulates PERK-eIF2α-ATF4 signalling in breast cancer.
Gupta A, Hossain MM, Miller N, Kerin M, Callagy G, Gupta S
Oncogene 2016 Nov 10;35(45):5860-5871
Oncogene 2016 Nov 10;35(45):5860-5871
A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.
Gaide Chevronnay HP, Janssens V, Van Der Smissen P, Liao XH, Abid Y, Nevo N, Antignac C, Refetoff S, Cherqui S, Pierreux CE, Courtoy PJ
Endocrinology 2015 Jun;156(6):2349-64
Endocrinology 2015 Jun;156(6):2349-64
PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response.
Gupta A, Hossain MM, Read DE, Hetz C, Samali A, Gupta S
Scientific reports 2015 Dec 17;5:18304
Scientific reports 2015 Dec 17;5:18304
Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC).
Caceres NE, Aerts M, Marquez B, Mingeot-Leclercq MP, Tulkens PM, Devreese B, Van Bambeke F
PloS one 2013;8(3):e58285
PloS one 2013;8(3):e58285
Proteomic analysis of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor complexes.
Kang MG, Nuriya M, Guo Y, Martindale KD, Lee DZ, Huganir RL
The Journal of biological chemistry 2012 Aug 17;287(34):28632-45
The Journal of biological chemistry 2012 Aug 17;287(34):28632-45
Ras inhibits endoplasmic reticulum stress in human cancer cells with amplified Myc.
Yaari-Stark S, Shaked M, Nevo-Caspi Y, Jacob-Hircsh J, Shamir R, Rechavi G, Kloog Y
International journal of cancer 2010 May 15;126(10):2268-81
International journal of cancer 2010 May 15;126(10):2268-81
Dysferlin overexpression in skeletal muscle produces a progressive myopathy.
Glover LE, Newton K, Krishnan G, Bronson R, Boyle A, Krivickas LS, Brown RH Jr
Annals of neurology 2010 Mar;67(3):384-93
Annals of neurology 2010 Mar;67(3):384-93
HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.
Huttunen HJ, Guénette SY, Peach C, Greco C, Xia W, Kim DY, Barren C, Tanzi RE, Kovacs DM
The Journal of biological chemistry 2007 Sep 21;282(38):28285-95
The Journal of biological chemistry 2007 Sep 21;282(38):28285-95
Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor.
Ohashi S, Koike K, Omori A, Ichinose S, Ohara S, Kobayashi S, Sato TA, Anzai K
The Journal of biological chemistry 2002 Oct 4;277(40):37804-10
The Journal of biological chemistry 2002 Oct 4;277(40):37804-10
Membrane topology of the murine fatty acid transport protein 1.
Lewis SE, Listenberger LL, Ory DS, Schaffer JE
The Journal of biological chemistry 2001 Oct 5;276(40):37042-50
The Journal of biological chemistry 2001 Oct 5;276(40):37042-50
Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes.
Mengesdorf T, Althausen S, Oberndorfer I, Paschen W
The Biochemical journal 2001 Jun 15;356(Pt 3):805-12
The Biochemical journal 2001 Jun 15;356(Pt 3):805-12
Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes.
Mengesdorf T, Althausen S, Oberndorfer I, Paschen W
The Biochemical journal 2001 Jun 15;356(Pt 3):805-12
The Biochemical journal 2001 Jun 15;356(Pt 3):805-12
Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.
Patterson CE, Schaub T, Coleman EJ, Davis EC
Molecular biology of the cell 2000 Nov;11(11):3925-35
Molecular biology of the cell 2000 Nov;11(11):3925-35
Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.
Patterson CE, Schaub T, Coleman EJ, Davis EC
Molecular biology of the cell 2000 Nov;11(11):3925-35
Molecular biology of the cell 2000 Nov;11(11):3925-35
Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.
Marmorstein AD, Marmorstein LY, Rayborn M, Wang X, Hollyfield JG, Petrukhin K
Proceedings of the National Academy of Sciences of the United States of America 2000 Nov 7;97(23):12758-63
Proceedings of the National Academy of Sciences of the United States of America 2000 Nov 7;97(23):12758-63
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Supportive validation
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- Western blot detection of GRP78 on rat liver lysate using Product # PA1-014A.
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- Western blot was performed using Anti-GRP78 Polyclonal Antibody (Product # PA1-014A) and a 72 kDa band corresponding to GRP78 was observed across cell lines and tissue extracts tested and increased upon Thapsigargin treatment. An uncharacterized band (*) at ~120 kDa was also observed in tissues . Whole cell extracts (30 µg lysate) of MCF-7 (Lane 1), MCF-7 treated with Thapsigargin (1uM for 24 Hours) (Lane 2), HeLa (Lane 3) and HeLa treated with Thapsigargin (1uM for 24 Hours) (Lane 4) and tissue extracts of Mouse Liver (Lane 5), Rat Liver (Lane 6) and Mouse Pancreas (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
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- Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in p19 Cells.
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- Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in HMVEC Cells.
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- Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in p19 Cells.
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- Immunofluorescent analysis of Grp78/BiP (green) in MDCK cells. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a Grp78/BiP rabbit polyclonal antibody (Product # PA1-014A), at a concentration of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-rabbit IgG Superclonal secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
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- Immunofluorescent analysis of Glucose Regulated Protein 78 using anti-Glucose Regulated Protein 78 polyclonal antibody (Product # PA1-014A) shows staining in Hela Cells.
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- Immunofluorescence analysis of GRP78 was performed using 70% confluent log phase MCF7 cells and MCF7 cells treated with 1 µM of Thapsigargin for 24 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with GRP78 Rabbit Polyclonal Antibody (Product # PA1-014A) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing strong Cytoplasmic localization. Panel e shows untreated cells with weak cytoplasmic signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
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- Figure 7 ER stress response evaluation. Immunoblot of Grp78 and actin as loading control in whole cell lysates from cells growing in standard conditions (line 1, WT cells in control medium; line 2, CIP-R cells in medium added by 0.2 mM ciprofloxacin), or in WT cells exposed for 24 h to 0.2 mM ciprofloxacin (line 3), or in cells exposed for 24 h to 0.5 ug/ml tunicamycin (TM; line 4, WT cells; line 5, CIP-R cells). The anti-Grp78 is a rabbit antiserum used at 1:500 dilution. The hatched square highlights the standard conditions of culture for each cell line.
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- Figure 5 Effect of miR-424 on UPR in HEK 293T cells. ( A ) Upper panel, shows a schematic representation of lentiviral vector used to generate miR-424 expressing clones. Lower panel, expression of GFP was monitored in 293T-control and 293T-miR-424 cells after treatment with (1.0 mug/ml) of doxycycline for 24 hours. ( B ) 293T-control and 293T-miR-424 cells were treated with (1.0 mug/ml) of doxycycline for 24 h and expression levels of miR-424 was quantified by qRT-PCR, normalizing against snoRNA. Error bars represent mean +- S.D. from two independent experiments performed in triplicate. *P < 0.05, two-tailed unpaired t-test comparing the expression in 293T-control and 293T-miR-424 cells. ( C ) 293T-control and 293T-miR-424 were transfected with the indicated UPR pathway reporter gene (ATF6, CHOP, XBP1) and 24 hours post transfection cells were treated with TG (1.0 muM). Normalized Firefly luciferase activities (Firefly/Renilla) relative to untreated control are shown. *P < 0.05, two-tailed unpaired t-test comparing the increase in luciferase activity in 293T-control and 293T-miR-424 cells. ( D ) 293T-control and 293T-miR-424 cells were either untreated or treated with (1.0 muM) TG for indicated time points and immunoblotting of total protein was performed using antibodies against ATF6, spliced XBP1, PERK, GRP78, phospho-eIF2alpha, total eIF2alpha and beta-actin. ( E ) Autorads obtained after probing the membrane with GRP78 antibody were analysed using Image J. Relative intens
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- Figure 2 Comparison of gefitinib resistance-related proteins in HCC827 and HCC827GR cells. ( a ) ER stress response elements (GRP78 (glucose regulated protein of a78 kDa molecular weight) and CHOP (CCAAT/enhancer-binding protein homologous protein)), beta-catenin, and cyclooxygenase-2 (COX-2). ( b ) Epithelial (E-cadherin) and mesenchymal (vimentin and N-cadherin) markers, as well as bypass signaling molecules (AXL and p-MET). ( c ) Cancer stem markers (CD133, CD44, and ABCG2). Whole-cell lysates were analyzed by Western blot for the proteins of interest, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The numbers under the bands show the relative densitometric ratios to the bands of the loading control.
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- Figure 8 Effect of anthocyanins from the flower petals of Hibiscus syriacus L. (Malvaceae, AHs) on ER stress and mtROS production. ( A ) and ( B ) The expression of GRP78, ATF4, p-eIF1alpha, CHOP, and beta-actin protein (left) and relative density (right). ( C ) The staining of MitoSOX Red and MitoTracker Green. ( D ) Survival rate of zebrafish. * p < 0.05 and *** p < 0.001 vs. UVB-irradiated cells and ### p < 0.001 vs. untreated cells (UT).
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- Figure 6. Effect of purified EDEM1/2/3 on M8B present in mCD3-delta-DeltaTM-HA. ( A ) ( a ) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing TAP-mCD3-delta-DeltaTM-HA was subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-HA antibody. ( b ) Eluate in ( a ) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-HA, anti-Myc, and anti-GRP78 (which is identical to BiP) antibodies. ( B ) ( a ) Eluate in ( A ) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. ( b ) N -glycans prepared from samples in ( a ) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.
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- Figure 7. Effect of purified EDEM1/2/3 on M8B present in ATF6alpha(C). ( A ) ( a ) Eluate obtained from EDEM1, 3-double knockout (DKO) cells overexpressing ATF6alpha(C)-TAP2 was subjected to SDS-PAGE under reducing conditions, silver-stained, and analyzed by immunoblotting using anti-Myc antibody. ( b ) Eluate in ( a ) was untreated (-) or treated (+) with EndoH, subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-Myc and anti-GRP78 (which is identical to BiP) antibodies. ( B ) ( a ) Eluate in ( A ) was incubated with purified EDEM1, EDEM2-TXNDC11 complex, or EDEM3(Q543N) for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. ( b ) N -glycans prepared from samples in ( a ) after 24 hr incubation were analyzed by mass spectrometry (MS). This experiment was conducted once.
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- Fig. 7 Irp2 deficiency impairs Fe-S cluster protein function and causes ER stress. a CRISPR/Cas9 depletion of Irp2 in INS-1 832/13 cells (sgIrp2.1, sgIrp2.2) and control parental cells grown in the presence of FAC (10 ug/ml), DFO (50 uM), or no treatment (NT) for 18 h. Irp2, Fth1, Ftl1 and TfR1 were assessed by western blot analysis to show efficacy of Irp2 knockdown and appropriate iron regulation. beta-Actin is a loading control. Asterisk, nonspecific band. b The total iron content measured by ICP-MS and normalized to total cellular protein ( n = 3 independent biological experiments). c , d Mitochondrial ( c ) and cytosolic ( d ) aconitase activity in control, sgIrp2.1, and sgIrp2.2 INS-1 cells grown in medium with or without supplemental FAC and normalized to total cellular protein. e , f Complex I activity ( e ) and complex IV activity ( f ) in lysates from control EV and shIrp2 cells grown in medium with or without supplemental FAC and normalized to total cellular protein ( n >= 3 independent biological experiments). g ATP production in control INS-1, sgIrp2.1, and sgIrp2.2 cells grown in medium with or without supplemental FAC and assayed under basal (5 mM) glucose and after stimulation with 15 mM glucose for 1 h. ATP production was normalized to total cellular protein ( n = 5 independent biological experiments). h Western blot analysis of eIF2alpha-P, eIF2alpha-total, and Grp78/BiP levels in sgIrp2.1 and sgIrp2.2 cells under basal glucose (5 mM) and after stimulation w
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- Figure 2 HRECs exposed to TNF-alpha, and high glucose for 24 h demonstrated increased GRP78 translocation. HRECs silenced for KDELR1 (KDEL-KD), confirmed with protein expression of KDELR1 in total cell lysates ( A ). GRP78 protein expression in cytosolic (Cyto) and membrane (Mem) fractions from various treatments. Na + /K + -ATPase served as an internal control for membrane fraction, while beta-tubulin represented a cytosolic fraction ( B ). Representative confocal immunofluorescence images of the cells stained for GRP78 and WGA ( C ). Data represent Mean +- SEM from 3 independent experiments performed in duplicates. ***p < 0.001; **p < 0.01; *p < 0.05, # p > 0.05.
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- Figure 7 HRECs exposed to TNF-alpha, and high glucose for 24 h demonstrated alterations in VE-Cadherin and GRP78 association. Whole cell lysate were immunoprecipitated with antibody against VE-Cadherin, and the immunocomplexes were analyzed by immunoblotting for GRP78 ( A ). Whole cell lysates were immunoprecipitated with complex partners (p-120 and beta-catenin) and immunoblotted with GRP78 ( B ). beta-tubulin served as an internal control. Data represent Mean +- SEM from 3 independent experiments performed in duplicates. ***p < 0.001; **p < 0.01.