Rabbit anti grp78

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-GRP78 is a primary antibody that specifically binds to the GRP78 (glucose-regulated protein 78) antigen. GRP78 is a member of the heat shock protein 70 (HSP70) family and plays a key role in the endoplasmic reticulum (ER) stress response. This antibody can be used in various immunoassays to detect and quantify GRP78 expression in biological samples.

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GRP78 (148 citations) Anti-GRP78 (45 citations) Rabbit polyclonal anti-GRP78 (2 citations) Rabbit monoclonal anti-GRP78 antibody (2 citations)

5 protocols using rabbit anti grp78

Western Blot Analysis of Brain Proteins

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Total brain tissues were sonicated using a LabSonic homogenizer (B. Braun Biotech Inc., Allentown, PA, USA), and the protein concentration in the brain samples was then quantified using a bicinchoninic acid assay kit (Pierce, CA, USA). Samples were then analyzed by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk containing 0.05% Tween in PBS. The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel). Membranes were then probed with secondary horseradish peroxidase-conjugated antibodies (1:3000; Amersham Biosciences, Little Chalfont, UK) for 1 h at room temperature. The blots were then visualized using chemiluminescent peroxidase substrate (ECL prime; GE Healthcare). Immunoreactivity for each protein band intensity was quantified using NIH ImageJ software [24 (link)] and normalized to β-actin as a loading control.

Lu J., Dou F, & Yu Z. (2019). The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease. Journal of Neuroinflammation, 16, 273.

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Immunofluorescence Staining of GRP78, Bungarotoxin, and Synaptophysin

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Antibodies used for immunofluorescence staining included rabbit anti-GRP78 (1:200, Cell Signaling, Danvers, MA, USA, Cat #C50B12), fluorophore 594-conjugated α-Bungarotoxin (1:1000, Thermo Fisher Scientific, Waltham, MA, USA, Cat #B13423) and rabbit anti-synaptophysin (1:200, Abcam, Cambridge, UK, Cat #ab32127).

Hyde L.F., Kong Y., Zhao L., Rao S.R., Wang J., Stone L., Njaa A., Collin G.B., Krebs M.P., Chang B., Fliesler S.J., Nishina P.M, & Naggert J.K. (2022). A Dpagt1 Missense Variant Causes Degenerative Retinopathy without Myasthenic Syndrome in Mice. International Journal of Molecular Sciences, 23(19), 12005.

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Macrophage Protein Expression Analysis

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Macrophages were lysed with Cell Lytic Buffer (Sigma) and the cell lysate was collected. For p47 ^phox estimation the cell lysate was centrifuged at 600g for 10 min at 4°C to remove unbroken cells and nuclei. The supernatant was then ultracentrifuged at 100,000g for 1hr at 4°C to isolate the membrane fraction. Protein was estimated by Bradford’s reagent and equal amounts of protein sample from each experimental condition was subjected to immunoblot analysis using the following primary antibodies: goat poly anti-MCPIP (1:500), rabbit anti-IRE-1 (1:500), rabbit anti-LC3 II (1:500), rabbit anti-C/EBPβ (1:100), rabbit anti-PPARγ (1:100), rabbit anti-FIZZ1 (1:500), monoclonal anti–p47^phox (1:200), and rabbit anti-Fas (1:1000; Santa Cruz Biotechnology); mouse polyclonal anti-GAPDH (1:1000); rabbit anti-GRP78 (1:500); rabbit anti-B ECLin 1(1:1000), goat anti-Arg1 (1:2000; Cell signaling). The immune complexes were detected autoradiographically using appropriate peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology) and enhanced chemiluminescence detection reagent ECL (GE Healthcare). Anti-β-actin and anti-GAPDH antibodies served as loading controls. Specific bands were quantified by densitometry using analytic software (Image J) and expressed as a ratio over loading controls.

Kapoor N., Niu J., Saad Y., Kumar S., Sirakova T., Becerra E., Li X, & Kolattukudy P.E. (2015). Transcription factors STAT6 and KLF4 implement macrophage polarization via the dual catalytic powers of MCPIP. Journal of immunology (Baltimore, Md. : 1950), 194(12), 6011-6023.

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Western Blot Analysis of Apoptotic Markers

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Western blot analysis was performed as described previously^{39 (link)} using the following antibodies: mouse anti-caspase-8 (Alexis Biochemicals, Grünberg, Germany), mouse rabbit anti-caspase-3, rabbit anti-caspase-9, mouse anti-CHOP, rabbit anti-GRP78, rabbit anti-PERK (all from Cell Signaling, Beverly, MA, USA), rabbit anti-DPAGT1 antibody (Abcam, Cambridge, MA, USA). Mouse anti-β-actin, mouse anti-vinculin (Sigma-Aldrich), or mouse anti-glyceraldehyde 3-phosphate dehydrogenase (HyTest, Turku, Finland) were used as loading controls. Goat anti-mouse immunoglobulin G (IgG), donkey anti-goat IgG, and goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.

Abhari B.A., McCarthy N., Le Berre M., Kilcoyne M., Joshi L., Agostinis P, & Fulda S. (2019). Smac mimetic suppresses tunicamycin-induced apoptosis via resolution of ER stress. Cell Death & Disease, 10(3), 155.

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ER Stress Induction in Cell Lines

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HEK293T and HeLa cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco), 50U/ml penicillin, 50 μg/ml streptomycin, in 5% CO₂ atmosphere at 37 °C. Plasmid transfection was carried out using LipofectAMINE (Invitrogen) according to manufacturer’s instructions. 2 μM Thapsigargin (Enzo Life Sciences) was used to induce ER stress. The primary antibodies used for western blot were rabbit anti-Grp78 (Cell Signaling Technology), mouse anti-Flag (Sigma), rabbit anti-GFP (Invitrogen) and mouse anti-β-Tubulin (CWBiotech).

Zhang P., Zhou L., Pei C., Lin X, & Yuan Z. (2016). Dysfunction of Wntless triggers the retrograde Golgi-to-ER transport of Wingless and induces ER stress. Scientific Reports, 6, 19418.

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