Bip grp78

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BiP/GRP78 is a molecular chaperone protein that plays a crucial role in the endoplasmic reticulum (ER) of eukaryotic cells. It is responsible for assisting in the proper folding and assembly of proteins within the ER.

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GRP78 (148 citations) Anti-GRP78/BiP (17 citations)

30 protocols using bip grp78

Molecular Markers of Endothelial Dysfunction

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Mice were sacrificed and heart, aorta and MRA were immediately harvested and frozen in liquid nitrogen and then stored at −80°C. Endothelial cells and tissue lysates were prepared as previously described.^{53 (link)} Western blot analysis for P-eNOS, T-eNOS, P-PERK, T-PERK, BiP(GRP78), P-eIF2-α, T-eIF2-α, CHOP, P-p38 and T-p38MAPK, P-ERK1/2 and T-ERK1/2 (1:1000 dilution, Cell Signaling Technology, Inc, USA), and ATF6, HuR or β-actin (1:500 dilution, Santa Cruz Biotechnology, Inc) was performed using specific antibodies as previously described.^{53 (link)}

Galán M., Kassan M., Kadowitz P.J., Trebak M., Belmadani S, & Matrougui K. (2014). Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction. Biochimica et biophysica acta, 1843(6), 1063-1075.

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Dissecting Apoptosis Signaling Pathways

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G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.

Vo D.K., Hartig R., Weinert S., Haybaeck J, & Nass N. (2019). G-Protein-Coupled Estrogen Receptor (GPER)-Specific Agonist G1 Induces ER Stress Leading to Cell Death in MCF-7 Cells. Biomolecules, 9(9), 503.

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Curcumin Modulates Cellular Stress Responses

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Curcumin, AO, MDC, NAC, z-VAD, CHX, and DCF-DA were purchased from Sigma (St. Louis, MO, USA). The class III PI3K inhibitor 3-MA was obtained from Calbiochem (La Jolla, CA, USA). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI, USA). Antibodies against PARP, caspase-3, p62, Beclin-1, CHOP, phospho-eIF2α (Ser51), IRE1-α, Bip/GRP78 and Nrf2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against LC3 (Sigma) were also used. The anti-β-actin, anti-ubiquitin, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Lee W.J., Chien M.H., Chow J.M., Chang J.L., Wen Y.C., Lin Y.W., Cheng C.W., Lai G.M., Hsiao M, & Lee L.M. (2015). Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress. Scientific Reports, 5, 10420.

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Protein Expression Analysis of Seipin and UPR

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PVAT and aortas tissues were homogenized in RIPA buffer, and the protein content was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) as previously described (8 (link)). Immunoblotting was performed using the antibodies against Seipin (Abnova, Taipei, Taiwan), Mac2 (Santa Cruz, CA, USA), BIP/GRP78, PDI, PERK, phospho-PERK (Thr980), eIF2a and phospho-eIF2a (Ser51) (Cell Signaling, Danver, MA, USA), and GAPDH (Millipore, Billerica, MA). The examined proteins were detected using an Odyssey V3.0 image scanning (Li-COR, Inc., Lincoln, NE, USA). The protein bands were analyzed using densitometry, and arbitrary densitometry units were quantified are expressed as mean ± SEM.

Wang M., Xing J., Liu M., Gao M., Liu Y., Li X., Hu L., Zhao X., Liao J., Liu G, & Dong J. (2021). Deletion of Seipin Attenuates Vascular Function and the Anticontractile Effect of Perivascular Adipose Tissue. Frontiers in Cardiovascular Medicine, 8, 706924.

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Antibody Characterization Protocol

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Antibodies against Bip/GRP78 (#3177), IRE1α (#3294), and β-actin (#3700) were obtained from Cell Signaling Technologies (Danvers, MA, USA). The CFTR antibody (#217 and #596) was from the Cystic Fibrosis Foundation Therapeutics (Bethesda, MD). The SGLT1 antibodies were from Invitrogen (PA5-88282, Waltham, MA, USA; for Western blot) and Abcam (ab14686, Waltham, MA, US; for immunofluorescence staining). The p-IRE1α antibody (#AP0878) was from ABclonal Technology (Woburn, MA, USA). The XBP1s antibody (#619,502) was from BioLegend (San Diego, CA, USA). The secondary antibodies were from LI-COR Biosciences (#D01216-10 and #D00226-05, Lincoln, NE, USA; for Western blot) and Jackson ImmunoResearch Laboratories (#147,158, West Grove, PA, USA; for immunofluorescence staining).

Sun Y., Zhang Y., Zhang J., Chen Y.E., Jin J.P., Zhang K., Mou H., Liang X, & Xu J. (2024). XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions. Cell & Bioscience, 14, 27.

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Evaluation of Autophagy Markers in Tg-Treated Cells

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Cells were harvested after 30 h of treatment with Tg, unless otherwise stated. Preparation of whole-cell lysates, SDS-PAGE, and immunoblot analysis were performed as described previously [33 (link)]. The following antibodies were used: alpha-tubulin (Abcam, ab7291), ATG5 (Cell Signaling Technology 2630), ATF4 (Cell Signaling Technology 11815), Bip/Grp78 (Cell Signaling Technology 3177), Caspase-3 (8G10) (Cell Signaling Technology 9665), Caspase-8 (1C12) (Cell Signaling Technology 9746), CHOP (Cell Signaling Technology 2895), Cleaved PARP (Asp214) (D64E10) (Cell Signaling Technology 5625), DR5 (Cell Signaling Technology 8074), FADD (G-4) (Santa Cruz sc-271748), FIP200 (Proteintech 17250–1-AP, GABARAP (Proteintech PM037), GABARAPL1 (Abcam ab86497), GABARAPL2 (Proteintech PM038), p-JNK (Cell Signaling Technology 9251), JNK (Cell Signaling Technology 9252), LC3B (Cell Signaling Technology 2775), PERK (Cell Signaling Technology 5683), XBP1s (BioLegend 647502).

Lindner P., Christensen S.B., Nissen P., Møller J.V, & Engedal N. (2020). Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components. Cell Communication and Signaling : CCS, 18, 12.

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Western Blot Analysis of ER Stress Markers

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Cells were lysed in RIPA lysis buffer, consisting of 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.8, 1% [w/v] Triton X-100, 1 mmol/L EDTA, 0.5 mmol/L phenyl-methanesulfonyl fluoride, 1× Complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN, USA) and a phosphatase inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Samples were centrifuged, and protein concentrations were measured by BCA assay (Thermo Scientific, Waltham, MA, USA). A total of 25μg denatured protein from tissues was separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane, the membrane was blocked with 5% nonfat milk solution and washed three times with buffer (Tris-buffered saline, 0.05% Tween). Western blotting was performed using antibodies recognizing proteins BiP/Grp78 (Cell Signaling, #3183), cleaved-ATF6 (Thermofisher, MA5–16172), CHOP (Cell Signaling, #2895) followed by species-appropriate secondary antibodies conjugated to HRP (BioRad). Proteins were visualized by enhanced chemiluminescence. Band intensities were quantified with the Image J software (National Institutes of Health, Bethesda, MD, USA).

Abdullahi A., Wang V., Auger C., Patsouris D., Amini-Nik S, & Jeschke M.G. (2020). CATECHOLAMINES INDUCE ENDOPLASMIC RETICULUM STRESS VIA BOTH ALPHA AND BETA RECEPTORS. Shock (Augusta, Ga.), 53(4), 476-484.

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Pancreatic Protein Expression Analysis

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The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557). The following primary antibodies were used for immunofluorescence: PITPNA (1:200, Sigma, SAB1400211). Antibodies were used on paraffin-embedded pancreata fixed in 4% paraformaldehyde for 3 h. Image densitometry of 16-bit TIF images for all western blots was performed using ImageJ. All original uncropped western blot images are presented in Supplementary Figs. 7–13.

Yeh Y.T., Sona C., Yan X., Li Y., Pathak A., McDermott M.I., Xie Z., Liu L., Arunagiri A., Wang Y., Cazenave-Gassiot A., Ghosh A., von Meyenn F., Kumarasamy S., Najjar S.M., Jia S., Wenk M.R., Traynor-Kaplan A., Arvan P., Barg S., Bankaitis V.A, & Poy M.N. (2023). Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction. Nature Communications, 14, 4250.

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Comprehensive Protein Extraction and Western Blot Analysis

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Total protein was extracted from 10 × 106 cells using M-PER mammalian extraction reagent supplemented with phosphatase/protease inhibitor cocktails (Thermo Fisher Scientific; Waltham, MA, USA), and the protein concentration was determined using a BCA Protein Assay (Thermo Fisher. Western blotting (WB) was performed as previously described [19 (link)]. In brief, equal amounts of cell lysate protein were separated on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Next, immunoblotting was performed: first with primary antibodies under gentle shaking overnight and then with the appropriate secondary antibody (HRP-coupled) for 2 h at room temperature. The immunoblotting results were visualized with a chemiluminescence detection system (LAS-3000 FUJIFILM). Primary antibodies against the following proteins were used: caspase-3, caspase-9, BIP/GRP78, and AIF (Cell Signaling Technology Europe-Bioke; Leiden, Nederland); BCL-2, β-actin, and α-tubulin (Santa Cruz Biotechnology-Bio-Connect); HPSPA1A (Abnova; Taipei, Taiwan); and HMGB1 (ABCAM; Cambridge, UK). Secondary antibodies (HRP-coupled) against rabbit-IgG and mousse-IgG were used (GE Healthcare).

Berehab M., Rouas R., Akl H., Duvillier H., Journe F., Fayyad-Kazan H., Ghanem G., Bron D., Lewalle P, & Merimi M. (2021). Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis. Cancers, 13(14), 3579.

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Western Blot Analysis of Protein Expression

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Lysates were prepared by suspending cells in Laemmli sample buffer (Sigma-Aldrich) and heated to 100 °C for 10 min, and resolved by SDS-PAGE (Bio-Rad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and primary antibodies were used for immunodetection: rabbit anti-AR (1:1000) [28 (link)] or mouse anti-actin (1:4000, Sigma-Aldrich) or rabbit anti-p-JNK, JNK, p-ERK, ERK, p-p38, p38 and Bip/GRP78 (1:1000, Cell signaling Technology, Inc., Danvers, MA, USA). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (1:5000, Millipore, Bedford, MA, USA), as well as the Western Blot Substrate kit (Bio-Rad) were used to detect chemiluminescence using a BioRad ChemiDoc™ XRS+ imaging system.

Chang K.C., Snow A., LaBarbera D.V, & Petrash J.M. (2014). Aldose Reductase Inhibition Alleviates Hyperglycemic Effects on Human Retinal Pigment Epithelial Cells. Chemico-biological interactions, 234, 254-260.

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