Jnk1 2

Manufactured by BD

Sourced in United States

JNK1/2 is a laboratory tool used in research applications. It functions as a kinase enzyme that is involved in cellular signaling pathways. This product is intended for use in scientific investigations, but a detailed description of its specific applications or intended use cannot be provided in an unbiased and factual manner.

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6 protocols using jnk1 2

Western Blot Analysis of Signaling Pathways

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WB analysis was performed on cell or tissue lysates that were separated by SDS/PAGE and transferred to nitrocellulose membranes. The primary antibodies and dilutions were as follows: beta actin (A5441, Sigma) at 1:5000, p Stat3 (#9145; Cell Signaling Technology) at 1:2000; Stat3 (#9139; Cell Signaling Technology) at 1:2000, ERK1/2 (#9107; Cell Signaling Technology) at 1:2000, pERK1/2 (#4370; Cell Signaling Technology) at 1:2000, p-H2AX (#9718, Cell Signaling Technology) at 1:1000, pAKT (#9271; Cell Signaling Technology) at 1:1000, AKT (sc-8312; Santa Cruz) at 1:1000, p-P38 (#9211; Cell Signaling Technology) at 1:1000, P38 (sc-535; Santa Cruz) at 1:200, pJNK (#9251; Cell Signaling Technology) at 1:1000, JNK1/2 (#554285; BD Pharmingen) at 1:500, pIKKα/β (#2697, Cell Signaling Technology) at 1:1000, IKKα (#136A Imgenex) at 1:1000, p-P65 (#3031, Cell Signaling Technology), P65 (c-20) (sc-372, Santa Cruz) at 1:1000.

Liang S., Ma H.Y., Zhong Z., Dhar D., Liu X., Xu J., Koyama Y., Nishio T., Karin D., Karin G., Mccubbin R., Zhang C., Hu R., Yang G., Chen L., Ganguly S., Lan T., Karin M., Kisseleva T, & Brenner D.A. (2018). NADPH Oxidase 1 in Liver Macrophages Promotes Inflammation and Tumor Development in Mice. Gastroenterology, 156(4), 1156-1172.e6.

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Immunoblot Analysis of Liver Signaling

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Liver extracts were prepared using Triton lysis buffer (20 mM Tris-pH 7.4, 1% Triton-X100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 1 μM sodium orthovanadate, 1 μM PMSF and 10 μg/mL leupeptin plus aprotinin). Extracts (30–50 μg of protein) were examined by immunoblot analysis by probing with antibodies to pSer⁶³-cJUN, pJNK, pMKK4, MKK4, pMKK7, and MKK7 (Cell Signaling Technologies), JNK1/2 (BD Pharmingen), cJUN and GAPDH (Santa Cruz) and β-Tubulin (Covance). Immunocomplexes were detected by fluorescence using anti-mouse and anti-rabbit secondary IRDye antibodies (Li-Cor) and quantitated using the Li-Cor Imaging system.

Vernia S., Cavanagh-Kyros J., Barrett T., Tournier C, & Davis R.J. (2016). Fibroblast growth factor 21 mediates glycemic regulation by hepatic JNK. Cell reports, 14(10), 2273-2280.

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Immunoblot Analysis of NF-κB Signaling Pathway

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For immunoblot analyses, cells were washed twice with PBS and lysed in lysis buffer consisting of 1% NP-40, 120 mM NaCl, 40 mM pH 7.4 Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined by Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Immunoprecipitation was performed as described previously [12 (link)].
Antibodies against the following proteins were purchased from the indicated sources: phospho-IKKα/β (2697), IKKα (2682), IKKβ (2370), phospho–NF-κB (3033), NF-κB (8242), phospho-IκB (2859), IκB (4814), phospho-JNK (4668), and K63-linkage Specific Polyubiquitin (5621) (Cell Signaling Technology, Danvers, MA, USA); JNK1/2 (554285; BD Biosciences, Franklin Lakes, NJ, USA); TRAF6 (sc-7221), GAPDH (sc-32233) and HSP90 (sc-13119) (Santa Cruz Biotechnology, Dallas, TX, USA); Rabbit IgG isotype control (02-6102) (Invitrogen, Waltham, MA, USA); Tubulin (T5109) (Sigma-Aldrich, St. Louis, MO, USA); IPMK (rabbit polyclonal antibody, raised against a mouse IPMK peptide corresponding to amino acids 295-311 (SKAYSTHTKLYAKKHQS; Covance) containing an added N-terminal cysteine) [8 (link)].

Lee H., Kim E, & Kim S. (2023). miRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling. Biomolecules, 13(2), 332.

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Protein Immunoblot Analysis of AKT and JNK

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Extracts (20–50 µg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT (Cell Signaling Technology 9272; RRID: AB_329827), pSer⁴⁷³ AKT (Cell Signaling Technology 9271; RRID: AB_329825), pThr³⁰⁸ AKT (Cell Signaling Technology 4056; RRID: AB_331163), JNK1/2 (BD Biosciences 554285, RRID: AB_395344), GAPDH (Santa Cruz Biotechnology sc-25778, RRID: AB_10167668), and αTubulin (clone B-5-1-2; Millipore-Sigma T5168; :RRID: 477579). IRDye 680LT-conjugated donkey anti-mouse immunoglobulin G (IgG) antibody (LI-COR Biosciences 926-68022, RRID: AB_10715072) and IRDye 800CW conjugated-goat anti-rabbit IgG (LI-COR Biosciences 926-32211, RRID: AB_621843) were used to detect and quantitate immune complexes with the Odyssey infrared imaging system (LI-COR Biosciences).

Han M.S., Perry R.J., Camporez J.P., Scherer P.E., Shulman G.I., Gao G, & Davis R.J. (2021). A feed-forward regulatory loop in adipose tissue promotes signaling by the hepatokine FGF21. Genes & Development, 35(1-2), 133-146.

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Western Blot Analysis of cJUN Signaling

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Tissue extracts were prepared using Triton lysis buffer (20 mM Tris-pH 7.4, 1% Triton-X100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF and 10 µg/mL leupeptin plus aprotinin). Extracts (30–50 µg of protein) were examined by immunoblot analysis by probing with antibodies to cJUN (Cell Signaling Technology Cat# 9165L, RRID:AB_2129578), pSer⁶³-cJUN (Cell Signaling Technology Cat# 9261S, RRID:AB_2130162), Flag M2 (Sigma-Aldrich Cat# F1804, RRID:AB_262044), GAPDH (Santa Cruz Biotechnology Cat# sc-365062, RRID:AB_10847862), JNK1/2 (BD Biosciences Cat# 554285, RRID:AB_395344), pThr¹⁸⁰-Pro-pTyr¹⁸²-JNK (pJNK) (Cell Signaling Technology Cat# 4668P, RRID:AB_10831195), NOVA1 (Abcam Cat# ab97368, RRID:AB_10680798), NOVA2 (Sigma-Aldrich Cat# AV40400, RRID:AB_1854572), and βTubulin (BioLegend Cat# 903401, RRID:AB_2565030). Immunocomplexes were detected by fluorescence using anti-mouse and anti-rabbit secondary IRDye antibodies (Li-Cor) and quantitated using the Li-Cor Imaging system

Vernia S., Edwards Y.J., Han M.S., Cavanagh-Kyros J., Barrett T., Kim J.K, & Davis R.J. (2016). An alternative splicing program promotes adipose tissue thermogenesis. eLife, 5, e17672.

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Protein Immunoblot Analysis of Cellular Signaling

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Extracts (20 to 50 µg of protein) were examined by protein immunoblot analysis by probing with antibodies to JUN (Cell Signaling Technology, Cat# 2315, RRID: AB_490780), pSer⁶³-JUN (Cell Signaling Technology, Cat# 9261, RRID: AB_213016292), GAPDH (Santa Cruz Biotechnology, Cat# sc-25778, RRID: AB_10167668), JNK1/2 (BD Biosciences, Cat# 554285, RRID: AB_395344), FLAG (M2) (MilliporeSigma, Cat# P2983, RRID: AB_439685), RXRα (Abcam, Cat# ab125001, RRID: AB_10975632), pSer²² mouse RXRα (affinity-purified rabbit polyclonal antibody made to keyhole limpet hemocyanine conjugated to Cys-QVNSSSLNpSPTGR), and α-tubulin (MilliporeSigma, clone B-5-1-2, Cat# T5168, RRID: _477579). IRDye 680RD-conjugated goat anti-mouse IgG antibody (LI-COR Biosciences, Cat# 926-68070, RRID: AB_10956588) and IRDye 800CW-conjugated goat anti-rabbit IgG (LI-COR Biosciences, Cat# 926-32211, RRID: AB_621843) were used to detect immune complexes with the Odyssey infrared imaging system (LI-COR Biosciences).

Vernia S., Lee A., Kennedy N.J., Han M.S., Isasa M., Cavanagh-Kyros J., Roy A., Syed A., Chaudhry S., Edwards Y.J., Gygi S.P., Gao G, & Davis R.J. (2022). Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome. Proceedings of the National Academy of Sciences of the United States of America, 119(44), e2210434119.

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