P c jun ser73

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P-c-Jun (Ser73) is an antibody that detects phosphorylation of the c-Jun protein at serine 73. This post-translational modification is involved in the regulation of c-Jun activity.

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C-Jun (263 citations) P-c-Jun (111 citations) P-JUN (10 citations) Anti‐p‐c‐JUN(ser73), (2 citations)

15 protocols using p c jun ser73

Cytokine and Signaling Pathway Analysis Protocol

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Recombinant human IL-1β, IL-6, IL-8, RANTES, and MMP-1 ELISA assays were purchased from R&D Systems (Minneapolis, MN). PGG was purchased from Sigma (G7548 >95% pure) for in vitro and from Cayman Chemicals (#16007) for animal study. Thiamet G (#13237, Cayman Chemicals), human cytokine antibody array C5 (AAH-CYT-5-2) and TRANCE ELISA (ELM-TRANCE-1) from RayBiotech (Norcross, GA), rat Cytokine/Chemokine Magnetic Bead Panel (RECYMAG65K27PMX; Sigma, MO), mouse O-GlcNAc (CTD110.6) (sc-59623), mouse A20 (#sc-166692), and mouse monoclonal β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Antibodies against p-TAK1 Thr^184/187 (#4531), p-JNK (#9251) p-JNK (#4511), p-c-Jun (Ser⁷³) (#9164), MyD88 (D80F5) (#4283), CYLD (D6O5O) (#12797) were purchased from Cell Signaling Technology (Beverly, MA). Anti-TAK1 (ab109526) and anti-TRAF6 (ab33915) antibodies were from Abcam (Cambridge, MA). Goat anti-rabbit and goat anti-mouse HRP-linked secondary antibodies were purchased from Cell Signaling Technology.

Umar S., Singh A.K., Chourasia M., Rasmussen S.M., Ruth J.H, & Ahmed S. (2022). Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model. Frontiers in Immunology, 13, 928436.

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Molecular Signaling Pathway Analysis

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Cisplatin, gentamicin, penicillin SP600125, and 3-methyladenine (3-MA) were purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Akt inhibitor II was purchased from Calbiochem (EMD Millipore, Darmstadt, Germany). Primary antibodies were used in the following dilutions: STAT1 at 1/1000, both pSTAT1-Ser727 and -Tyr701 at 1/500, both p STAT3-Ser727 and -Tyr705 at 1/500, Akt at 1/1000, pAkt-Ser473 at 1/1000, p-c-Jun (Ser73) at 1/500, c-Jun at 1/1000, LC3 at 1/1000 (all from Cell Signaling, Bioconcept, Allschwil, Switzerland), STAT3 at 1/1000 (Santa Cruz Biotechnology, Labforce AG, Nunningen, Switzerland), Beclin-1 at 1/1000 (Novus Biologicals, Littleton, MA, USA), and GAPDH at 1/5000 (Abcam, Labforce AG, Nunningen, Switzerland).

Levano S, & Bodmer D. (2015). Loss of STAT1 protects hair cells from ototoxicity through modulation of STAT3, c-Jun, Akt, and autophagy factors. Cell Death & Disease, 6(12), e2019-.

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Investigating Neuroprotective Effects of GK

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GK was extracted and separated by Jiangsu Kanion Modern Traditional Chinese Medicine Research Institute with 98% purity. SH-SY5Y cells were purchased from Cell Bank of the Chinese Academy of Sciences (no. CRL-2266) which is imported from the ATCC (Shanghai, China). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Bestbio Biotechnology (Shanghai, China). The ROS assay kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit antibodies against p38, p-p38 (Thr180/Tyr182), JNK, p-JNK (Thr183/Tyr185), p53, p-p53 (Ser15), c-Jun, p-c-Jun (Ser73), Bcl-2, cleaved caspase-3, caspase-3, tubulin, actin and the secondary antibody were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit antibodies against Bax and cleaved caspase-9 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PVDF membrane and ECL western detection reagent were obtained from Bio-Rad Laboratory (Hercules, CA, USA). All other reagents were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) unless otherwise stated.

Liu Q., Li X., Li L., Xu Z., Zhou J, & Xiao W. (2018). Ginkgolide K protects SH-SY5Y cells against oxygen-glucose deprivation-induced injury by inhibiting the p38 and JNK signaling pathways. Molecular Medicine Reports, 18(3), 3185-3192.

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Ceramide and S1P Signaling Inhibitors

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ABC294640 was purchased from DC Chemicals (Shanghai, China). SKI-II (4-[[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol) was purchased from Tocris Bioscience (Ellisville, Mo). Cell permeable short-chain C6 ceramide was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). S1P was purchased from Cayman Chemical Co. (Ann Arbor, MI). 5-fluorouracil (5-FU) and cisplatin were purchased from Sigma (Shanghai, China). SP600125 and JNK inhibitor II (JNKi-II) were obtained from Selleck (Shanghai, China). Antibodies against phospho (p)-AKT (Ser 473), p-AKT (Thr 308), p-ribosomal protein S6 kinase 1 (S6K1) (Thr-389), p-JNK1/2 (Thr 183/Tyr 185) and p-c-Jun (Ser73) were purchased form Cell Signaling Tech (Denver MA). Anti-AKT1, SphK2, c-Jun and tubulin antibodies were obtained from Santa Cruz (Santa Cruz, CA).

Xun C., Chen M.B., Qi L., Tie-Ning Z., Peng X., Ning L., Zhi-Xiao C, & Li-Wei W. (2015). Targeting sphingosine kinase 2 (SphK2) by ABC294640 inhibits colorectal cancer cell growth in vitro and in vivo. Journal of Experimental & Clinical Cancer Research : CR, 34(1), 94.

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Immunofluorescence Staining of Tissue Sections

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IF staining was performed as described previously (51 (link), 52 (link)). After blocking, the slides were incubated with the primary antibodies to Ln-γ2 (#ab210959, Abcam; dilution ratio, 1:500), pan-cytokeratin (#ab7753, Abcam; dilution ratio, 1:400), α-SMA (#19245, Cell Signaling Technology; dilution ratio, 1:200), CD3 (#ab1669, Abcam; dilution ratio, 1:150 dilution), CD4 (#ab183685, Abcam; dilution ratio, 1:200 dilution), CD8 (#ab217344, Abcam; dilution ratio, 1:200 dilution), EpCAM (#36746, Cell Signaling Technology; dilution ratio, 1:100), p-c-Jun (Ser⁷³) (#3270, Cell Signaling Technology; dilution ratio, 1:800), and p-c-Fos (Ser³²) (#5348, Cell Signaling Technology; dilution ratio, 1:200) at 4°C overnight in a moist chamber. After thorough washing, the slides were then incubated with Alexa Fluor 594– or 488–conjugated secondary antibodies (Invitrogen). Last, all slides were mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; #S36938, Thermo Fisher Scientific). Images were captured with an OLYMPUS FV2000 fluorescence microscope.

Li L., Wei J.R., Dong J., Lin Q.G., Tang H., Jia Y.X., Tan W., Chen Q.Y., Zeng T.T., Xing S., Qin Y.R., Zhu Y.H., Li Y, & Guan X.Y. (2021). Laminin γ2–mediating T cell exclusion attenuates response to anti–PD-1 therapy. Science Advances, 7(6), eabc8346.

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Western Blot Analysis of Cell Signaling Proteins

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Cells were lysed in NP-40 lysis buffer (Novex/Life Technologies) and a cocktail of protease inhibitors (cOmplete ULTRA Tablets, Roche) and Pefabloc SC PLUS (Roche). The insoluble material was sedimented by centrifugation (12,000g, 10 min, 4 °C), and the supernatants were collected and frozen at −80 °C. Protein content was determined using a BCA Protein Assay kit (Pierce, Rockford, IL). Equivalent amounts of protein were electrophoresed on 4–12% Bis-Tris gels (Life Technologies) and transferred to polyvinylidene difluoride membranes (Life Technologies). Membranes were blocked and incubated with primary antibodies (1:1,000; β-catenin: #610154, BD Biosciences; GAPDH: #ab37168, Abcam; β-actin: #13E5; LRP6, #2560; pLRP6, #S1490; SAPK/JNK: #9252S; pSAPK/JNK (Thr183/Tyr185), #9255S; cJUN: #9165S; pcJUN (Ser73) #9164S, Cell Signaling Technology, Inc., Danvers, MA) overnight, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibodies (#7074S and #7076S, 1:1,000, Cell Signaling Technology). The protein bands were visualized with ImmunoStar Western C (Bio-Rad, Hercules, CA) and the ChemiDoc XRS+ System (Bio-Rad).

Movérare-Skrtic S., Henning P., Liu X., Nagano K., Saito H., Börjesson A.E., Sjögren K., Windahl S.H., Farman H., Kindlund B., Engdahl C., Koskela A., Zhang F.P., Eriksson E.E., Zaman F., Hammarstedt A., Isaksson H., Bally M., Kassem A., Lindholm C., Sandberg O., Aspenberg P., Sävendahl L., Feng J.Q., Tuckermann J., Tuukkanen J., Poutanen M., Baron R., Lerner U.H., Gori F, & Ohlsson C. (2014). Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures. Nature medicine, 20(11), 1279-1288.

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Phytochemical Compound Procurement for Inflammation Study

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EGCG (≥95% purity HPLC), EGC (≥95% HPLC), and EC (≥90% HPLC) compounds were purchased from Sigma (St. Louis, MO; cat# E4143, E3768, E7153). Gelatin from bovine skin was purchased from Sigma (G6650). Cyclooxygenase (Cox)-1 and Cox-2 antibodies were purchased from Cayman Chemical (Ann Arbor, MI; cat# aa160110 and aa 570–598). P-P38, p-JNK, and p-ERK, p-TAK1 (Thr184/187), p-cJun(Ser73), and p65-NF-κB were purchased from Cell Signaling Technologies (Danvers, MA; Cat# 4511, 9251,4695,4508,3270, and 3033). β-Actin and Lamin A/C loading controls were purchased from Santa Cruz (Santa Cruz, CA; sc-47,778; sc-6215).

Fechtner S., Singh A., Chourasia M, & Ahmed S. (2017). Molecular insights into the differences in anti-inflammatory activities of green tea catechins on IL-1β signaling in rheumatoid arthritis synovial fibroblasts. Toxicology and applied pharmacology, 329, 112-120.

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Chel A Compound Extraction and Analysis

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Chel A was isolated from Goniothalamus cheliensis by the Kunming Institute of Botany, Chinese Academy of Sciences (Kunming, Yunnan, China) as previously described (1 (link), 3 ). The Chemicals cycloheximide (CHX) and MG132 were purchased from Calbiochem (San Diego, CA, USA). Luciferase assay substrate and EGF were from Promega (Madison, WI, USA). The antibodies specific against c-Jun, c-Jun(D), p-c-Jun Ser63, p-c-Jun Ser73, p-AKT Ser473, p-AKT Thr308, AKT, p-Erk1/2, Erk1/2, p-p38, p38, p-JNK1/2, JNK1/2, PARP, cleaved PARP, Caspase-3, cleaved Caspase-3, p53, p-p53 Ser15, p-Chk1 Ser345, Chk1, GFP, GAPDH, were purchased from Cell Signaling Technology (Beverly, MA, USA). HA antibody was obtained from Covance Inc. (Princeton, NJ, USA). Antibodies specific against PHLPP1 and PHLPP2 were purchased from Bethyl Laboratories (Montgomery, TX, USA). Antibodies against β-Actin and α-Tubulin were bought from Sigma (St. Louis, MO, USA). The plasmid, HA-PHLPP1 and HA-PHLPP2 were from Addgene (Cambridge, MA, USA). The plasmids, AP-1-luciferase reporter, dominant negative c-Jun mutant plasmid TAM67, and GFP-c-Jun were used and described in our previously studies(13 (link)–15 (link)).

Zhu J., Zhang J., Huang H., Li J., Yu Y., Jin H., Li Y., Deng X., Gao J., Zhao Q, & Huang C. (2014). Crucial Role of C-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin A (Chel A) Inhibition of Cell Transformation. Cancer prevention research (Philadelphia, Pa.), 7(12), 1270-1281.

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Molecular Regulation of c-Jun and JNK

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The dominant-negative mutant of c-Jun (TAM67) [19 (link)] and the dominant-negative mutant of JNK (DN-JNK) [20 (link)] have been described in our previous studies [19 (link),20 (link)]. The PRL-TK-luciferase expression vector and pSUPER vector were purchased from Promega and have been used in our previous studies [21 (link)]. Adenovirus-driven GFP (Ad-GFP) and Adenovirus-driven-GFP–p27^Kip1 (Ad-GFP–p27) plasmids were constructed and have been used in our previous studies [18 (link)]. Constructs expressing Egfr promoter-driven luciferase reporter and its various deletions as indicated in Figure 2(D) were a gift from Dr D.T. Smoot (Department of Medicine and Cancer Center, Howard University, Washington, DC, U.S.A.). Antibodies against c-Jun, EGFR, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), histone H3, JNK1/2, p-c-Jun Ser⁶³, p-c-Jun Ser⁷³ and p-JNK were purchased from Cell Signaling Technology. Antibodies against c-Fos, Fra-1, SP1 (specificity protein 1) and c-Myc were from Santa Cruz Biotechnology. The antibody against p27^Kip1 was obtained from Abcom Biochemicals.

Fang Y., Wang Y., Wang Y., Meng Y., Zhu J., Jin H., Li J., Zhang D., Yu Y., Wu X.R, & Huang C. (2014). A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition. Biochemical Journal, 463(Pt 3), 383-392.

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RANTES and MMP Regulation in Fibrosis

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Recombinant human RANTES/CCL5, Met-RANTES and IL-1β, and MMP-1 ELISA Duoset were purchased from R&D systems (Minneapolis, MN, USA). MMP-13 ELISA was purchased from Ray Biotech (Norcross, GA, USA). MMP-1 and MMP-13 SYBR Green primers were purchased from Qaigen (Valencia, CA, USA). SMARTpool ON-TARGETplus RANTES small-interfering RNA (siRNA) and On-target plus non targeting siRNA (D-001810-10) control were purchased from GE Dharmacon (Lafayette, CO, USA). MMP-1, MMP-13, type I collagen, and lamin A/C antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Rabbit monoclonal or polyclonal antibodies against p-p38 (Thr¹⁸⁰/Tyr¹⁸²), p-ERK (Thr²⁰²/Tyr²⁰⁴), p-JNK(Thr¹⁸³/Tyr¹⁸⁵), p-PKCδ (Thr⁵⁰⁵), total p-38, total JNK, total ERK, p-c-Jun (Ser⁷³), and p-ATF-2 (Thr^69/71) were from Cell Signaling Technologies (Beverly, MA, USA). Rabbit polyclonal antibodies against CCR1 and CCR5 were from BioVision (Milpitas, CA) and GeneTex (Irvine, CA, USA), respectively. Heparinase III (flavobacterium heparinum derived), heparan sulfate degrading lyase was purchased from Sigma (St. Louis, MO, USA). type I collagen was purchased from Advanced Biometrix (Carlsbad, CA, USA). Inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB 02190), PKCδ (Rottlerin), and NF-κB (PDTC) were purchased from EMD Millipore (Billerica, MA, USA).

Agere S.A., Akhtar N., Watson J.M, & Ahmed S. (2017). RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts. Frontiers in Immunology, 8, 1341.

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