Mouse anti chop

Manufactured by Cell Signaling Technology

Sourced in United States

Mouse anti-CHOP is an antibody product developed by Cell Signaling Technology. It is designed to detect the CHOP protein, which is a transcription factor involved in the cellular stress response.

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14 protocols using mouse anti chop

Protein Extraction and Quantification from Brain Tissue

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Brains were dissected immediately following euthanization. One hemisphere was then homogenized in protein lysis buffer (PLB: 50 mM Tris pH 6.8, 150 mM NaCl, 20 mM EDTA, 1 mM EGTA, 0.5% SDS, 0.5% NP40, sarkosyl 0.5%, 1 mM prefabloc, 10 mM orthovanadate, 2.5 mM sodium fluoride) [37] (link), [38] (link). Total protein concentrations were then determined using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL). Brain homogenates and lysates were both stored in aliquots at −20°C.
Freshly thawed brain homogenates and cell lysates were each diluted to 15 µg/µL and loaded onto either 8.5 or 10% SDS PAGE gels, transferred to 0.45 µm PVDF membranes (Immobilon-P, Millipore, Billeria, MA) and probed with the following primary antibodies: mouse anti-FLAG (#F3165 Sigma Aldrich, St. Louis, MO), mouse anti-ubiquilin (clone 3D5E2, Invitrogen, Carlsbad, CA) which recognizes ubiquilin proteins, rat anti-BiP (sc-13539 Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-actin (Santa Cruz Biotechnology), rabbit anti-PDI (#2446 Cell Signaling, Danvers, MA), mouse anti-CHOP (#2895 Cell Signaling). Secondary antibodies used were horse radish peroxidase conjugated goat anti-mouse (#31430), goat anti-rabbit (#31460), goat anti-rat (NA935V GE Healthcare) and rabbit anti-goat (#31492 Thermo Scientific).

Safren N., El Ayadi A., Chang L., Terrillion C.E., Gould T.D., Boehning D.F, & Monteiro M.J. (2014). Ubiquilin-1 Overexpression Increases the Lifespan and Delays Accumulation of Huntingtin Aggregates in the R6/2 Mouse Model of Huntington's Disease. PLoS ONE, 9(1), e87513.

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Comprehensive Antibody Panel for Autophagy and UPR Analysis

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Rabbit anti-LC3B (#3868), rabbit anti-ATG5 (#12994), rabbit anti-ATG7 (#8558), rabbit anti-Beclin-1 (#3495), rabbit anti-ATG16L1 (#8089), rabbit anti-BIP (#3177), rabbit anti-ATF6 (#65880), rabbit anti-ATF4(#11815), rabbit anti-XBP1 (#12782), rabbit anti-PERK (#5683), rabbit anti-IRE1 (#3294), rabbit anti-p-eif2α (#3398), rabbit anti-FIP200 (#12436), and mouse anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (dilution concentration 1:1000). Rabbit anti-p-IRE1 (PA1-16927) was purchased from Invitrogen (Waltham, MA, USA) (dilution concentration 1:1000). Mouse anti-p62 (18420-1-AP), mouse anti-HA-tag (66006-2-Ig), and mouse anti-β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China) (dilution concentration 1:5000). HRP-conjugated goat anti-mouse (G1214) and goat anti-rabbit (G1213) were purchased from Servicebio (Wuhan City, China) (dilution concentration 2:5000). Alexa Fluor 568 goat anti-mouse IgG, IgM (H + L) (A-11004) and Alexa Fluor 647 goat anti-mouse-IgG (H + L) (A-21445) were purchased from Thermo Fisher (Waltham, MA, USA) (dilution concentration 1:500). Immunofluorescence antibodies were diluted in PBS. Immunoblot antibodies were diluted in TBST.

Su W.Q., Yu X.J, & Zhou C.M. (2021). SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response. Viruses, 13(12), 2467.

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Western Blot Analysis of CHOP Expression

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To evaluate CHOP expression, nuclear and cytosolic fractions were prepared from BMM using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Proteinase inhibitor cocktail tablets (EDTA-free) (Roche) were added to the buffers. For immunoblotting, cytoplasmic and nuclear fractions were diluted with 5× sample buffer (Thermo Fisher Scientific) and 10× reducing agent (Thermo Fisher Scientific), then incubated in boiling water for 5 min. Proteins were separated on 4–20% Tris/Glycine gels (BioRad) and immunoblotting was performed as described^{32 (link)}. The following primary antibodies were used: mouse anti-CHOP (Cell Signaling Technology, #2895), rabbit anti-Lamin B1 (Abcam, #ab16048), and mouse anti-GAPDH (Santa Cruz Biotechnologies, #sc-32233). HRP-conjugated secondary antibodies (R&D Systems, #HAF008, #HAF007) against their respective primary antibodies were incubated with membranes for 1 h at room temperature. Proteins were visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

Navid F., Gill T., Fones L., Allbritton-King J.D., Zhou K., Shen I., Van Doorn J., LiCausi F., Cougnoux A., Randazzo D., Brooks S.R, & Colbert R.A. (2024). CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis. Scientific Reports, 14, 12293.

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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 Cochlear Frozen Sections

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Cochleae were sampled and fixed as previously mentioned. For immunofluorescence staining of frozen sections, the tissues were air-dried and treated with 1% Triton in PBS for 5 min, followed by incubation in blocking solution for 15 min. Then, the tissues were incubated with primary antibodies overnight at 4 °C. On the second day, the tissues were washed with PBS three times and incubated with Alexa Fluor anti-mouse/rabbit secondary antibody for 3 h. After PBS washes, the tissues were mounted with Vectorshield. Images were taken with a Zeiss 780 confocal microscope. The primary antibodies used for immunofluorescence were as follows: monoclonal anti-acetyl tubulin (1:200, Sigma, T7451, USA), rabbit anti-myosin VIIa (1:500, Proteus BioSciences, Ramona, CA, USA), rabbit anti-spectrin α (1:200, D8B7, Abcam, Cambridge, UK), anti-ATP8A2 (1:200, provided by Dr. Xianjun Zhu as shown previously [6 (link)]), TMEM30A monoclonal antibody (supernatant 1:3, a gift from Dr. Robert Molday, University of British Columbia, Canada), and mouse anti-CHOP (1:200, Cell Signaling Technology, USA). Nuclei were counterstained with either propidium iodide (PI) or DAPI (1:500).

Xing Y., Peng K., Yi Q., Yu D., Shi H., Yang G, & Yin S. (2023). TMEM30A is essential for hair cell polarity maintenance in postnatal mouse cochlea. Cellular & Molecular Biology Letters, 28, 23.

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Immunofluorescence Analysis of Apoptosis Markers

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Hep3B cells were seeded in a confocal dish and received the indicated treatment for 24 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min and blocked with 1% BSA for 1 h. Then, the samples were incubated with primary antibodies for 18 h at 4 °C, followed by incubating with secondary antibodies at 37 °C in the dark. Cellular nuclei were counterstained with Hoechst 33342 (Invitrogen, Waltham, MA, USA). Images were captured through laser confocal microscopy (Leica, Deer Park, IL, USA). The primary antibodies including rabbit anti-Bax (#41162) and mouse anti-CHOP (#2895) were purchased from Cell Signaling Technology, and rabbit anti-Bip (ab21685) was purchased from abcam.

Li M., Duan F., Pan Z., Liu X., Lu W., Liang C., Fang Z., Peng P, & Jia D. (2023). Astragalus Polysaccharide Promotes Doxorubicin-Induced Apoptosis by Reducing O-GlcNAcylation in Hepatocellular Carcinoma. Cells, 12(6), 866.

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Immunoblot Analysis of PGA2-Induced Apoptosis

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For immunoblot analysis, proteins were extracted from PGA₂-treated cells. Cells were lysed in RIPA buffer (Cell signaling Technology, Boston, MA, USA) containing cocktails of protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (FIVE photon Biochemicals, San Diego, CA, USA). All antibodies were commercially available as follows. Rabbit anti-cleaved PARP1 (c-PARP1), anti-ATF3, anti-ATF4, anti-DR5 (death receptor 5), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, and mouse anti-CHOP were purchased from Cell Signaling Technology (Boston, MA, USA). Chicken anti-GAPDH antibodies were obtained from Merck Millipore Korea (Seoul, Korea), respectively. Peroxidase-conjugated antibodies (HRP-conjugated anti-rabbit or -mouse IgG) were from Sigma-Aldrich Inc., and KPL (Gaithersburg, MD, USA, HRP-conjugated anti-chicken IgG).

Park K.M., Park J.Y., Pyo J., Lee S.Y, & Kim H.S. (2022). Induction of DR5-Dependent Apoptosis by PGA2 through ATF4-CHOP Pathway. Molecules, 27(12), 3804.

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Immunoblot Analysis for Protein Expression

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Immunoblot was performed as previously described (34 (link)). The cell lysates were subjected to 10% to 12% SDS-PAGE. Primary antibodies used in this study were as follows: mouse anti-GRP78 (BiP, 1:2000, BD Transduction Laboratories #610979), mouse anti-GAPDH (1:2000, Santa Cruz #sc-32233), mouse anti-CHOP (1:1000, Cell Signaling #2895), rabbit anti-cleaved-PARP (1:1000, Cell Signaling #5625), and rabbit anti-cleaved-Caspase 7 (1:1000, Cell Signaling #8438). Secondary antibodies used were as follows: HRP-conjugated goat anti-mouse (1:5000, Santa Cruz #sc-516102), HRP-conjugated goat anti-rabbit (1:3000, Santa Cruz #sc-2357). The band quantification was determined using the Bio-Rad Image Lab 6.0.1 software.

Yamamoto V., Wang B, & Lee A.S. (2023). Suppression of head and neck cancer cell survival and cisplatin resistance by GRP78 small molecule inhibitor YUM70. Frontiers in Oncology, 12, 1044699.

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Quantification of Podocyte Proteins

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The proteins from tissues or cells were fractionated by electrophoresis on 10% SDS-PAGE and electro-blotted to polyvinylidene difluoride filter membranes and incubated with the primary antibody at 4°C, and then with a HRP-conjugated secondary antibody. Primary antibodies: rabbit anti-APLNR, goat anti-nephrin (sc-32529; Santa Cruz Biotechnology), rabbit anti-synapodocin (sc-50459; Santa Cruz Biotechnology), rabbit anti-podocin (sc-21009; Santa Cruz Biotechnology), rabbit anti-zonula occludens-1 (ZO-1) (sc-8146; Santa Cruz Biotechnology), rabbit anti-Wilm’s tumor protein (WT1) (sc-192; Santa Cruz Biotechnology), mouse anti-Ub (MAB1510; Millipore), rabbit anti-k48-linkage specific polyubiqutinated protein (k48, 8081; Cell Signaling Technology, Danvers, MA, USA), mouse anti-CHOP (2895; Cell Signaling Technology) and rabbit anti-phospho-eIF2α (3398; Cell Signaling Technology). Densitometry was performed with Image J software (NIH, USA). To verify equal loading, antibody to GAPDH was used.

Guo C., Liu Y., Zhao W., Wei S., Zhang X., Wang W, & Zeng X. (2015). Apelin promotes diabetic nephropathy by inducing podocyte dysfunction via inhibiting proteasome activities. Journal of Cellular and Molecular Medicine, 19(9), 2273-2285.

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Western Blot Analysis of Neuroinflammatory Markers

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Mechanically pulverized frozen striatum samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Neta Scientific) containing 1:100 protease inhibitor cocktail (MilliporeSigma), centrifuged for 30 minutes at 12,000 g at 4°C, and protein concentrations were assessed using a BCA assay (ThermoFisher). SDS-PAGE was performed on 10% SDS tris-glycine gels using 20 μg protein per lane (BioRad). The membranes were blocked using EveryBlot Blocking Buffer (BioRad) for 5 minutes, followed by primary antibody incubation overnight at 4°C in EveryBlot at 1:1000 concentration using the following antibodies: mouse anti-NFκB (Cell Signaling Cat. 8242S, Lot: 16), rabbit anti-ATF4 (Cell Signaling Cat. 11815S, Lot: 4), rabbit anti-eIF2α (Cell Signaling Cat. 5324S, Lot: 6), mouse anti-CHOP (Cell Signaling Cat. 2895S, Lot: 13), mouse anti-4-HNE (R&D Systems Cat. MAB3249, Lot: WXN0521121), and mouse anti-β-actin (MilliporeSigma Cat. A5316, Lot: D0615). Membranes were incubated in anti-mouse or anti-rabbit horse radish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) at 1:5000 in 0.1% TBS-T for 1 hour at room temperature. Enhanced chemiluminescence was performed using ProSignal Pico detection reagent (Genesee Scientific).

Atone J., Wagner K., Koike S., Yang J., Hwang S.H, & Hammock B.D. (2023). Inhibition of Soluble Epoxide Hydrolase Reduces Paraquat Neurotoxicity in Rodents. Environmental toxicology and pharmacology, 98, 104070.

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