Rabbit anti phospho jnk

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phospho-JNK is a primary antibody that specifically recognizes the phosphorylated forms of c-Jun N-terminal kinase (JNK), an important signaling molecule involved in various cellular processes. This antibody can be used to detect and quantify the activation of JNK in biological samples.

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21 protocols using rabbit anti phospho jnk

Western Blotting Analysis of Cardiac Signaling

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Western blotting analysis was performed as described previously [13 (link)]. Mouse hearts or neonatal rat cardiomyocytes (NRCMs) were lysed with lysis buffer (20 mM Tris-HCl, pH 7.4; 1% Triton X-100; 1 mM EDTA; 30 mM HEPES; 50 mM Na₄P₂O₇; 100 mM NaF) containing 1× Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) and phosphatase inhibitors. Proteins were resolved by SDS-PAGE and electrotransferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). The antibodies against rabbit anti-Akt (#9272), rabbit anti-Akt2 (#2962), rabbit anti-phospho-p70S6K (#9204), rabbit anti-p70S6K (#9202), rabbit anti-phospho-S6 ribosomal protein (#2211), rabbit anti-S6 ribosomal protein (#2217), rabbit anti-phospho-ERK (#9101), rabbit anti-ERK (#9102), rabbit anti-phospho-JNK (#9251), rabbit anti-JNK (#9252), rabbit anti-GAPDH (#2118) (Cell Signaling Technology), goat anti-ZO-1 (sc-8146), mouse anti-Akt1 (sc-5298) (Santa Cruz Biotechnology), mouse anti-Cx43 (BD Biosciences, 610062) were used. Immunoreactive proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Fremont, CA, USA). Densitometric quantitation was achieved using Image J software (NIH).

Ock S., Lee W.S., Kim H.M., Park K.S., Kim Y.K., Kook H., Park W.J., Lee T.J., Abel E.D, & Kim J. (2018). Connexin43 and zonula occludens-1 are targets of Akt in cardiomyocytes that correlate with cardiac contractile dysfunction in Akt deficient hearts. Biochimica et biophysica acta. Molecular basis of disease, 1864(4 Pt A), 1183-1191.

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Protein Expression Analysis by Western Blot

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Protein extraction, Western blotting, and densitometric quantification were performed as described [51 (link)]. The following primary antibodies were used: rabbit anti-phospho-JNK (#9251, 1:1000; Cell Signaling Technology), rabbit anti-phospho-p38 (#9215, 1:1000; Cell Signaling Technology), and mouse anti-actin (MAB1501, 1:10,000; Merck Millipore, Billerica, MA, USA). Mouse anti-rabbit (sc-2357; 1:10,000; Santa Cruz Biotechnology) and horse anti-mouse (#7076, 1:3000; Cell Signaling Technology) were used as secondary antibodies. Original, uncropped blots are shown in Figure S3.

Seitz T., Hackl C., Freese K., Dietrich P., Mahli A., Thasler R.M., Thasler W.E., Lang S.A., Bosserhoff A.K, & Hellerbrand C. (2021). Xanthohumol, a Prenylated Chalcone Derived from Hops, Inhibits Growth and Metastasis of Melanoma Cells. Cancers, 13(3), 511.

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Immunoblotting of Signaling Pathways

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THP1 cells were lyzed in 1X SDS-PAGE loading buffer with protease and phosphatase inhibitor cocktails (Roche). Samples were resolved on a 4%−20% gradient SDS-PAGE gel. Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S). Western blots were incubated with respective HRP-conjugated secondary antibodies and visualized using ECL reagents (Pierce).

John S.P., Sun J., Carlson R.J., Cao B., Bradfield C.J., Song J., Smelkinson M, & Fraser I.D. (2018). IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages. Cell reports, 25(1), 95-106.e6.

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Western Blot for Protein Analysis

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Lens cell lysate samples were mixed with Laemmli buffer (Sigma-Aldrich) and were heated to 95° C for 10 minutes. After resolution with Mini Protean TGX Precast gel 4–15%, proteins from gels were transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The following primary antibodies were used for immunodetection: mouse anti-human AR (1:1000; Santa Cruz, TX, USA), mouse anti-actin (1:1000; Sigma-Aldrich). For detection of extracellular signal-related kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK), we used rabbit anti-phospho ERK1/2 (1:1000; Cell Signaling, MA, USA), rabbit anti-ERK1/2 (1:1000; Cell Signaling), rabbit anti-phospho JNK (1:1000; Cell Signaling), and rabbit anti-JNK (1:1000; Cell Signaling), respectively. Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (1:2000; Millipore, Bedford, MA, USA), as well as the Immun-Star WesternC Kit (Bio-Rad, CA, USA) were used to detect chemilluminescence using a BioRad ChemiDoc XRS+ imaging system.

Snow A., Shieh B., Chang K.C., Pal A., Lenhart P., Ammar D., Ruzycki P., Palla S., Reddy G.B, & Petrash J.M. (2014). Aldose reductase expression as a risk factor for cataract. Chemico-biological interactions, 234, 247-253.

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Immunofluorescence Imaging of Adipose Tissue

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Immunofluorescence was performed on 5–8 μm cryostat sections of tissues freshly embedded in OCT as described^{29 (link)}. The sections were fixed in 4% paraformaldehyde in PBS and then stained with rat anti-PECAM (1:200, BD Biosciences #557355), mouse anti-α smooth muscle actin (1:200, Abcam #ab7817), rabbit anti-phospho-JNK (1:200, Cell Signaling), rabbit anti-UCP1 (1:200, Abcam #ab10983) and rabbit anti-ERα (1:200, Abcam #ab2746) as described^{29 (link)}. Secondary antibodies, used at a 1:500 dilution, included cy3 donkey anti-rabbit, cy3 donkey anti-mouse, cy3 donkey anti-rat, cy5 donkey anti-rat, cy5 donkey anti-rabbit, cy3 donkey anti-mouse were from Jackson ImmunoResearch. Immunostaining images were collected on a Zeiss LSM500 confocal microscope, an Olympus IX70 inverted microscope or an Olympus upright BX40 microscope. Direct GFP and RFP fluorescence for whole-adipose depots were imaged and photographed with a Zeiss Stemi SV11 microscope. Cryostat sectioning was performed with a Microm HM505 E cryostat. For BrdU staining, the cells, sections or tissues were fixed and washed in H₂O, incubated with 1 N HCl at 37 °C for 45 min, washed in H₂O, incubated in 0.1 M NaBO₄ for 10 min and subjected to immunohistochemistry.

Lapid K., Lim A., Clegg D.J., Zeve D, & Graff J.M. (2014). Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells. Nature communications, 5, 5196.

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Western Blot Analysis of Protein Signaling

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After treatment, protein extracts for total cellular fractions were isolated in a cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) supplemented with 0.1 mg PMSF and a 1/100 dilution of protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Scraped samples were then centrifuged at 10000 rpm for 15 min at 4°C. The supernatants were used for Western blotting. Protein concentrations were measured using the Bio-Rad protein dye microassay (Bio-Rad, Hercules, USA). Proteins were separated on a 12% SDS-PAGE gel and then transferred onto PVDF membranes. The membrane was blocked with a solution of TBS and 5% fat-free milk for 1 h, then incubated overnight with rabbit anti-A-FABP, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-TLR4 (all from Cell Signaling Technology, Beverly, MA, USA), or monoclonal mouse anti-β-actin (Santa Cruz Biotechnology, Heidelberg, Germany). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG in TBS for 2 h at room temperature. The membrane was then exposed to film before development.

Li H., Luo H.Y., Liu Q., Xiao Y., Tang L., Zhong F., Huang G., Xu J.M., Xu A.M., Zhou Z.G, & Dai R.P. (2018). Intermittent High Glucose Exacerbates A-FABP Activation and Inflammatory Response through TLR4-JNK Signaling in THP-1 Cells. Journal of Immunology Research, 2018, 1319272.

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Protein Extraction and Western Blotting Protocol

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Protein extraction and Western blotting were performed as described^{31 (link)} using the following primary antibodies: goat anti-FGF9 (AF-273-NA, 1:2,000; R&D Systems), rabbit anti-phospho-ERK (#9101, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-JNK (#9251, 1:1000; Cell Signaling Technology) and mouse anti-actin (MAB1501, 1:10,000; Merck Millipore, Billerica, MA, USA). Donkey anti-goat (sc-2020; 1:1,000; Santa Cruz Biotechnology, Heidelberg, Germany), chicken anti-rabbit (sc-2955; 1:10,000; Santa Cruz Biotechnology) and horse anti-mouse (Santa Cruz Biotechnology, sc-2005, 1:3,000) were used as secondary antibodies.

Seitz T., Freese K., Dietrich P., Thasler W.E., Bosserhoff A, & Hellerbrand C. (2020). Fibroblast Growth Factor 9 is expressed by activated hepatic stellate cells and promotes progression of hepatocellular carcinoma. Scientific Reports, 10, 4546.

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Cisplatin-Induced Apoptosis Pathway

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Cisplatin (CDDP) was purchased from Sigma‐Aldrich (St Louis, MO, USA). JNK inhibitor SP600125">SP600125 was purchased from Sigma‐Aldrich and dissolved in DMSO. Antibodies used for western blotting were rabbit anti–ANXA3 (Saierbio, Tianjing, China), rabbit anti–phospho‐JNK (Cell Signaling Technology), rabbit anti–survivin (Abcam, Cambridge, MA, USA), mouse anti–caspase 8 (Cell Signaling Technology), rabbit anti–caspase 3 (Cell Signaling Technology) and mouse anti–β‐actin (Sigma‐Aldrich). The secondary antibodies coupled to HRP were purchased from ZSGB‐BIO (Beijing, China). Recombinant Human ANXA3 was purchased from R&D (Minneapolis, MN, USA). ANXA3 neutralizing antibody was purchased from Sigma‐Aldrich.

Wang L., Li X., Ren Y., Geng H., Zhang Q., Cao L., Meng Z., Wu X., Xu M, & Xu K. (2019). Cancer‐associated fibroblasts contribute to cisplatin resistance by modulating ANXA3 in lung cancer cells. Cancer Science, 110(5), 1609-1620.

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Immunoblotting and Immunofluorescence Antibodies

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The following antibodies were used in immunoblotting and immunofluorescence experiments: rabbit anti-human p75^NTR (1:1,000; G3231; Promega), rabbit anti-TrkA (1:1,000; Millipore), rabbit anti-phosphoTyr674/5 (1:1,000; Cell Signaling), mouse anti-HA (1:2,000; Sigma-Aldrich), mouse anti-FLAG M2 (1:1,000; Sigma-Aldrich), mouse anti β-actin (1:1,000; Sigma-Aldrich), rabbit MBP-probe (1:1,000; Santa Cruz), rabbit anti-Cleaved Caspase-3 (1:1,000, 9661S; Cell Signaling), rabbit anti phospho-p38 (1:1,000, 9211; Cell Signaling), rabbit anti p38 (1:1,000, 9212; Cell Signaling), rabbit anti JNK (1:1,000, 9252; Cell Signaling), rabbit anti phospho-JNK (1:1,000, 9251; Cell Signaling), goat anti-choline acetyltransferase (1:200, AB144P; Millipore), rabbit anti Cy3 (1:500; Jackson), goat anti mouse Ig/HRP (1:10,000; Jackson), goat anti rabbit Ig/HRP (1:10,000; Jackson), goat IRDye800 (1:15,000; Rockland), and goat anti-mouse antibodies coupled to either Alexa 555 or Alexa 488 (Invitrogen). The DNA was stained with DAPI (1:1,000).

Franco M.L., García-Carpio I., Comaposada-Baró R., Escribano-Saiz J.J., Chávez-Gutiérrez L, & Vilar M. (2021). TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition. Life Science Alliance, 4(4), e202000844.

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Investigating Inflammatory Signaling Pathways

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Aze was purchased from Selleckchem (Houston, TX, USA). SP600125 was obtained from Calbiochem (Darmstadt, Germany). LPS (Escherichia coli, serotype 011:B4), mouse anti-β-actin (1:2000, cat# A5316) and rabbit anti-lamin B1 (1:2000, cat# SAB1306342) antibodies, dimethyl sulfoxide (DMSO), and adenylyl-imidodiphosphate (AMPPNP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (IgG) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Thermo Scientific (Rockford, IL, USA). Antibiotic-antimycotic reagent, Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). The rabbit anti-phospho-IκBα (1:1000, cat# ab97783) antibody was supplied by Abcam (Cambridge, MA, USA). Rabbit anti-phospho-JNK (1:1000, cat# 4668), rabbit anti-JNK3 (1:1000, cat# 2305), rabbit anti-phospho-c-Jun (1:1000, cat# 9261), rabbit anti-c-Jun (1:1000, cat# 9165), rabbit anti-iNOS (1:1000, cat# 2982), rabbit anti-COX2 (1:1000, cat# 4842), mouse anti-NF-κB p65 (1:1000, cat# 6956), mouse anti-IκBα (1:1000, cat# 4814) antibodies, and horseradish peroxidase-conjugated anti-rabbit (1:2000, cat# 7074) and anti-mouse IgG (1:2000, cat# 7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Nguyen P.L., Bui B.P., Duong M.T., Lee K., Ahn H.C, & Cho J. (2021). Suppression of LPS-Induced Inflammation and Cell Migration by Azelastine through Inhibition of JNK/NF-κB Pathway in BV2 Microglial Cells. International Journal of Molecular Sciences, 22(16), 9061.

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