Immunoblotting was performed as described previously [4 (link),19 (link)]. The primary antibodies were used as follows: transforming growth factor-beta1 (TGF-β1, #ab179695, Abcam), connective-tissue growth factor (CTGF, #ab6992, Abcam), pro-interleukin-1beta (pro-IL-1β, #ab9722, Abcam), pro-IL-18 (#ab191860, Abcam), NOD-like receptor pyrin domain-containing protein 3 (NLRP3, #ab214185, Abcam), tumor necrosis factor-alpha (TNF-α, #ab205587), inducible nitric oxide synthase (iNOS, #ab178945), superoxide dismutase-2 (SOD2/MnSOD, #ab13534, Abcam), thioredoxin (TRX, #ab109385), thioredoxin-interacting protein (TXNIP, #ab188865), Parkin (#2132, Cell Signaling Technology), succinate dehydrogenase complex subunit A (SDHA, #ab66484, Abcam), C/EBP homologous protein (CHOP, L63F7, #2895, Cell Signaling), inositol-requiring protein-1α (IRE-1α, phospho S724, #ab37073, Abcam), B-cell lymphoma-2 (Bcl-2, #ab196495, Abcam), Bcl2-associated X (Bax, #ab32503, Abcam), p-JNK (#ab76572), p-MEK4 (#4514, Cell Signaling), p-ASK1 (#ab278547), p-P38 (#ab4822), p-MEK3 (#ab79586), beta actin (β-actin, #ab8226, Abcam). Images were analyzed with an image analyzer (Odyssey® CL Imaging System, LI-COR Biosciences, NE, USA). Optical densities were obtained using the sham group as 100% reference and normalized with β-actin.
Chop l63f7
Manufactured by Cell Signaling Technology
Sourced in United States
CHOP (L63F7) is a mouse monoclonal antibody that recognizes the C/EBP homologous protein (CHOP). CHOP is a transcription factor that is induced in response to various cellular stresses, including endoplasmic reticulum (ER) stress. The antibody can be used for the detection of CHOP in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Ab191860, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Odyssey cl imaging system, by LI COR (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Spa801, by Enzo Life Sciences (1 mentions)
Spa 826, by Enzo Life Sciences (1 mentions)
Spa 810, by Enzo Life Sciences (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
α stat3, by Cell Signaling Technology (1 mentions)
α tubulin monoclonal antibody, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Beta actin ab8227, by Abcam (1 mentions)
Lumiglo substrate, by Cell Signaling Technology (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Atf3 c19, by Santa Cruz Biotechnology (1 mentions)
Hybond, by Cytiva (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
6 protocols using chop l63f7
1
Immunoblotting for Oxidative Stress Markers
2
Protein Extraction and Western Blot Analysis
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Protein was extracted from islets or MIN6 cells with radioimmunoassay precipitation buffer (ThermoFisher) and quantified using bicinchoninic acid assay (ThermoFisher). 15–30 μg protein was resolved on a pre-cast 10% or 12% NuPAGE Bis-Tris gel (ThermoFisher), and transferred to a PVDF membrane as previously described [26 (link),30 (link)]. These were blocked for 1 h in 5% skim-milk, and probed at 4 °C overnight with antibodies purchased from: Sigma–Aldrich, Creb3l2 (HPA015068), beta actin (A5441); tubulin, (T6074), SEC23a, (ABC424); ThermoFisher, O-linked N-Acetylglucosamine (MAI-072); Santa Cruz, Pan 14-3-3 (sc-1657); and Cell Signalling Technology, CHOP (L63F7) (CST2895), phospho-EIF2a (Ser51) (CST 9721), phospho-mTOR (Ser2481) (CST 2974), phospho-PERK (Thr980) (CST 3179), phospho-ACC (ser79) (CST 3661), and cleaved caspase-3 (CST 9662). After chemiluminescent detection, quantification was carried out using ImageJ (NIH, Bethesda, MA, USA). All quantitative data was corrected for loading, as determined in the same gels using antibodies against beta actin, Pan 14-3-3, or tubulin.
3
Antibody Acquisition for HSP and TRX Analysis
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Antibodies against heat shock protein 25 (HSP25, SPA-801), heat shock protein 60 (HSP60, SPA-806), heat shock protein 70 (HSP70, SPA-810), heat shock protein 90 (HSP90, SPA-835), glucose-regulated protein 78 (GRP78, SPA-826), and glucose-regulated protein 75 (GRP75, SPA-825) were purchased from Enzo Life Sciences Inc., (Farmingdale, NY, USA). The antibody against cytosolic thioredoxin-1 (TRX-1, ATRX-06) was purchased from IMCO Corp (Stockholm, Sweden), thioredoxin-interacting protein (TxNiP, K0205-3) from MBL (Medical and Biological Laboratories Co. Ltd, Nagoya, Japan), and 4-hydroxy-2-nonenal (4-HNE, HNE11-S) from Alpha Diagnostic IntI Inc. (San Antonio, TX, USA). Antibodies against CCAAT/enhancer-binding protein homologous protein (CHOP, L63F7) and protein disulfide isomerase (PDI, C81H6) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against GAPDH (sc-25778) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
4
Phosphoprotein Analysis in Liver Lysates
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Liver lysates from mice were analyzed by SDS PAGE followed by immunoprobing with antibodies against phosphoSmad2 (Ser465/467-1:1000), phosphoStat3 (Y705-1:1000), phosphoNF-κB (S536-1:500) and CHOP (L63F7-1:1000) (Cell Signaling). To normalize per total proteins loaded, proteins were analyzed on parallel gels and probed with polyclonal antibodies against α-tubulin monoclonal antibody, α-Smad2, α-Stat3 and α-NF-κB (Cell Signaling). Blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG or antimouse IgG antibody (GE Healthcare Life Sciences, Chicago, IL) and detected by Enhanced Chemiluminiscent Substrate (Thermo Scientific, Rockford, IL).
5
Protein Expression Analysis Protocol
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Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 30 μL/mL aprotinin. The proteins (20–30 μg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare) and blocked with 5% BSA. Primary antibodies (1:1000 dilution) included NRF2 (D1Z9C), BiP/GRP78 (C50B12) and CHOP (L63F7) from Cell Signaling; beta-actin (ab8227) from Abcam and ATF3 (C19) from Santa Cruz. After secondary antibody incubation (1:3000, 2 h), the proteins were detected using Lumiglo substrate (Cell Signaling Technology, CA).
6
Immunofluorescence Assay for Cardiac p-TAK1 and CHOP
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The heart tissues were embedded in OCT compound and cut approximately 5 µm thick sections. For immunofluorescence, slides were permeated with 0.5% Triton X‐100 for 10 minutes and blocked with goat serum at room temperature for 1 hour. Then, they were incubated with an antibody targeting anti‐p‐TAK1 (1:100, Cell Signaling Technology). The slides were washed and followed by a further incubation at room temperature for 1 hour with an AlexaFluor®488 Goat anti‐Rabbit IgG (H+L) (cat# ab150077, Abcam) at 2 µg/mL and Phalloidin‐iFluor 594 reagent (1:100, cat# ab176757, Abcam). Nuclear DNA was labelled in blue with DAPI. Cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA in 0.1%PBS‐Tween for 1 hour. The cells were then incubated with CHOP (L63F7) (1:100, cat# 2895, Cell Signaling Technology) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with an AlexaFluor®488 Goat anti‐Mouse secondary (cat# ab150117, Abcam) at 2 µg/mL. Nuclear DNA was labelled in blue with DAPI. The results were calculated blindly by counting the positive staining cells in 10 high‐power field of vision (HPF)/sections.
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