Nobiletin, Z-VAD-FMK, Z-DEVD-FMK, dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) protease inhibitor cocktail, salubrinal, rabbit anti-human β-actin antibodies, and Dulbecco’s modified Eagle′s medium (DMEM) were purchased from Sigma (St. Louis, MO, USA). An annexin V-FITC/PI apoptosis detection kit was purchased from Pharmingen (San Diego, CA, USA). Antibodies against pro-caspase 9, cleaved caspase 9, caspase 8, pro-caspase3, cleaved caspase 3, Mcl-1, Bax, Bcl-xl, Bad, p-Bad, PARP-1, Bcl-2, 14-3-3, PDI, GRP78, calreticulin, cytochrome C, p-PERK, PERK, p-eIF2α, eIF2 α, ATF6-f, ATF4, IRE-1α, CHOP, p-JNK, p-c-jun, JNK, p-JNK, MAPKp38, p-MAPKp38, ERK, p-ERK, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Cell Signaling Technology (Danvers, MA, USA). Fetal bovine serum (FBS) was obtained from Biowest (Nuaillé, France).
P jnk
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany, Canada, Japan
P-JNK is a laboratory reagent used to detect and quantify the phosphorylated form of the c-Jun N-terminal kinase (JNK) protein. JNK is a key signaling molecule involved in various cellular processes such as cell growth, differentiation, and apoptosis. The phosphorylated form of JNK, or P-JNK, serves as a marker for the activation of the JNK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Cell Signaling Technology (401 mentions)
P akt, by Cell Signaling Technology (155 mentions)
P erk1 2, by Cell Signaling Technology (153 mentions)
β actin, by Cell Signaling Technology (151 mentions)
Gapdh, by Cell Signaling Technology (111 mentions)
Erk1 2, by Cell Signaling Technology (99 mentions)
β actin, by Santa Cruz Biotechnology (74 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (70 mentions)
Bcl 2, by Cell Signaling Technology (69 mentions)
P iκbα, by Cell Signaling Technology (67 mentions)
Cleaved caspase 3, by Cell Signaling Technology (65 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (55 mentions)
Caspase 3, by Cell Signaling Technology (55 mentions)
Pvdf membrane, by Merck Group (50 mentions)
P p38 mapk, by Cell Signaling Technology (46 mentions)
P c jun, by Cell Signaling Technology (43 mentions)
P stat3, by Cell Signaling Technology (42 mentions)
β actin, by Merck Group (38 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (38 mentions)
Nf κb p65, by Cell Signaling Technology (37 mentions)
925 protocols using p jnk
1
Molecular Mechanisms of Apoptosis Regulation
2
Osteosarcoma Cell Lines: Culture and Drug Treatments
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Human osteosarcoma cell lines MG-63, Saos-2 and U-2 OS were purchased from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China) and cultured in McCoy’s 5a medium (Gibco, Los Angeles, CA) containing 10% fetal bovine serum (Gibco) and ampicillin and streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. All cell lines were used within 20 passages. The antibody against HSP90AA1was obtained from Proteintech. The antibodies against LC3, p62, cleaved PARP, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, p38, p-p38 and actin were obtained from Cell Signaling Technology. Cisplatin, doxorubicin, methotrexate, bafilomycin A1, rapamycin, LY294002 and 3-methyladenine were purchased from Sigma Aldrich (St. Louis, MO, USA).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from BMMs and C3H10 cells and from the bones of mice used in the OVX experiment using a radioimmunoprecipitation assay lysis buffer (RIPA) (Sigma-Aldrich), and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (EMD Millipore, Bedford, MA, USA). Protein equivalents were separated using 10% SDS PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked for an hour at room temperature with 5% skim milk in Tris-buffered saline with Tween 20. After incubating primary antibodies overnight at 4 °C, the membranes were nurtured with corresponding HRP conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA.) The bands were visualized employing ECL Luminous Liquid (EMD Millipore). ImageJ software was used to compute the relative grey level of proteins (NIH). Anti-TRAIL was procured from Santa Cruz Bio (Santa Cruz, CA, USA). Anti-CTSK, TRAP, NFATc1, OPG, RUNX2, OCN, RANKL, COL1a, p-P65, P65, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-IKBα, IKBα, IKKα, IKKβ, and p-IKKα/β were all purchased from Cell Signaling Technology (Danvers, MA, USA).
4
Osteoarthritis Chondrocyte Protein Analysis
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Total lysates of human OA chondrocytes were prepared using RIPA buffer (Biosesang, Gyeonggi-do, Korea). Primary antibodies against MMP3, MMP13 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), COL2A1 (1:1000 dilution, Abcam), ACAN (1:500 dilution, Invitrogen), p38, p-p38, ERK, p-ERK, JNK, p-JNK, p65, p-p65, IκBα, p-IκBα, or β-actin (1:1000 dilution, Cell Signaling Technology) and appropriate HRP-conjugated secondary antibodies (1:2000 dilution, Bethyl) were used. Enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific) was used to detect for specific bands. Chemiluminescent signals were analyzed on a ChemiDoc XRS gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA), and intensity of specific bands was quantified using Image J software (NIH, MD, USA).
5
Evaluating SNX-2112 Cytotoxicity and Signaling
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SNX-2112 was synthesized as previously described in our lab with >98.0% purity [42 (link)], dissolved in Dimethyl sulfoxide (DMSO) to obtain a 100 mM stock solution, and stored at −20°C. TRAIL was purchased from Merck Millipore (Waltham, MA, USA). N-Acetyl-cysteine (NAC) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Bafilomycin A1 (BFA) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for GAPDH, Bcl-2, Bcl-xL, FLIP, pro-caspase 3, cleaved-caspase 3 (c-caspase 3), cleaved-caspase 8 (c-caspase 8), cleaved-PARP (c-PARP), Akt, p-Akt (Ser473), DR4, DR5, LC3, Beclin1, Atg7, p62, p-mTOR, p-S6, p-4EBP1, p53, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).
6
Comprehensive Western Blot Analysis of Signaling Proteins
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Western blot was performed as described previously (9 (link)). Samples (20 μg) were run on a 10% SDS-PAGE gel followed by blotting to a nitrocellulose membrane. Membranes were blocked and incubated with the following antibodies: TRPV4 (ACC-034, Alomone Labs), SMAD3 (9523, Cell Signaling Technology), p-SMAD 3 (9520, Cell Signaling Technology), ERK (4695, Cell Signaling Technology), p-ERK (4370, Cell Signaling Technology), JNK (9252s, Cell Signaling Technology), p-JNK (4671s, Cell Signaling Technology), P38 (9212s, Cell Signaling Technology), p-P38 (4511s, Cell Signaling Technology), AKT (4691,Cell Signaling Technology), p-AKT (4060, Cell Signaling Technology), STAT3 (9132, Cell Signaling Technology), and p-STAT3 (9131, Cell Signaling Technology). Corresponding secondary antibodies conjugated to horseradish peroxidase were used for detection. Staining was detected using chemiluminescence and quantified by Image Lab software (Bio-Rad). All expression data was provided relative to GAPDH (diluted 1:500, Aspen) staining for the same samples on the same gels.
7
Hsp90 Isoform-Specific Inhibitor Synthesis
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The inhibitors for specific Hsp90 isoforms [KUNA115 and NDNA1065 (Hsp90α), KUNB106 and its analog, NDNB1151 (Hsp90β), KUNG65 (Grp94), T1, T2, and T3 (TRAP1)] were synthesized and purified by the laboratory of Dr. Brian Blagg at the University of Notre Dame [(Crowley et al., 2017 (link); Mishra et al., 2021a (link); Mishra et al., 2021b (link); Merfeld et al., 2023 (link)), Figure 1 ]. Lipopolysaccharide (LPS, from E. coli O111:B4), 17-AAG and penicillin:streptomycin (tissue culture) were purchased from VWR (Radnor, PA, United States). Dulbecco’s Modified Eagle Medium (DMEM), trypsin-EDTA, fetal bovine serum (FBS), and G418 disulfate solution were obtained from Corning (Corning, NY, United States). Griess reagents for nitric oxide assay (sodium nitrite, sulfanilamide, N-1-naphthylethylenediamine) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies used for Western blots include goat anti-rabbit IgG-horseradish peroxidase, and goat anti-mouse IgG-horseradish peroxidase (Invitrogen, Waltham, MA, United States); p-ERK, t-ERK, p-p38, t-p38, p-JNK, and t-JNK (Cell Signaling, Beverly, MA, United States).
8
Proteasome and Apoptosis Pathway Analysis
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The proteins were extracted from lung tissue or cells and lysed by using RIPA buffer (high; cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice for >30 min. Protein levels were quantified using BCA reagent. Samples were loaded 10 µl per lane and electrophoresed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4˚C overnight. The primary antibodies used were: 20S proteasome β1 (1:500; cat. no. sc-374405; Santa Cruz Biotechnology, Inc.), iNOS (1:1,000; cat. no. AF0199; Affinity Biosciences), cleaved caspase 3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. ET1609-42; HUABIO, Inc.), total JNK Antibody (1:500; cat. no. ET1601-28; HUABIO, Inc.) and GAPDH (1:5,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.). The secondary antibodies used were horseradish peroxide-conjugated goat anti-mouse IgG (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.). The bands were visualized using the Chemiluminescent Substrate kit (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.) combined with a Bio-Rad exposure system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ 1.53e.
9
Signaling Pathway Protein Antibodies
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TRAF6, p-TAKThr184/187, p-IRAK4Thr345/Ser346, IRAK4, p-P38, P38, p-JNK, JNK, p-ERK, ERK, GR, and NF-κBp65 antibodies were purchased from Cell Signaling Technologies (Danvers, MA) with respective catalog numbers 8028S, 90C7, D6D7, 4363, 4511S, 8690S, 9251S,9252T, 4370S, 4695S, 12041T, D14E12. p-TAKSer439 was obtained from Abcam (Cat EPR2863). β-Actin and Lamin B antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA;, sc-47778, sc-6217). β-tubulin was purchased Sigma (St. Louis, MO cat# T8328). All antibodies were diluted in 5% BSA/TBS-T according to manufactures recommendation. JWH-015 was sourced from Tocris (Cat# 1341; ≥99% HPLC) and dissolved in DMSO at a stock concentration of 10 mM. For in vivo studies, JWH-015 was dissolved 3% DMSO/PBS.
10
IFN-γ and TNF-α Modulate HaCaT Cell Signaling
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Human keratinocyte HaCaT cells were purchased from Elabscience (Catalog No. EP-CL-0090, Houston, TX, United States). The cells were cultured in a high-glucose-containing Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, United States) supplemented with 1% penicillin/streptomycin (Gibco-BRL, Gaithersburg, MD, United States) and heat-inactivated 10% fetal bovine serum (Gibco-BRL) in a humidified incubator containing 5% CO2 at 37°C. Recombinant human IFN-γ, TNF-α, Alexa 594 goat anti-rabbit antibody (cat.no. A-11037), Alexa 488 goat anti-rabbit antibody (cat.no. A-11034), and Alexa 488 goat anti-mouse antibody (cat.no. A-10680) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). DRAQ5™, p-IκBα (cat.no. 2859), IκBα (cat.no. 9242), p65 (cat.no. 8242), β-actin (cat.no. 3700), p-JNK (cat.no. 9251), JNK (cat.no. 9252), p-ERK (cat.no. 9101), ERK (cat.no. 9102), p-p38 (cat.no. 9211), p38 (cat.no. 9212), p-STAT1 (Tyr) (cat.no. 9167), p-STAT1 (Ser) (cat.no. 9177), and STAT1 (cat.no. 9172) antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). PCNA (cat.no. sc-56), p50 (cat.no. sc-8414), secondary horseradish peroxidase (HRP)–conjugated anti-mouse (cat.no. sc-2357), and anti-rabbit (cat.no. sc-516102) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).
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