Phospho tak1

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-TAK1 is a primary antibody that detects the phosphorylated form of TAK1 (Transforming growth factor-beta-activated kinase 1). TAK1 is a serine/threonine protein kinase that plays a pivotal role in various cellular signaling pathways.

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Lab products found in correlation

Spelling variants of this product

Rabbit anti-phospho-Tak1 (3 citations) Phospho-TAK1 (Thr184/187) (3 citations) Phospho(p)-Tak1 (2 citations) Anti-phospho-TAK1 (2 citations)

14 protocols using phospho tak1

Celastrol Sensitizes Cancer Cells to Cisplatin

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Cisplatin and Celastrol (>98% pure) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and trypsin solution (EDTA) were bought from Gibco (Invitrogen, Grand Island, NY). Antibodies against NF-κB p65 (rabbit, monoclonal), phospho-NF-κB p65 (rabbit, monoclonal), TAK1 (rabbit, monoclonal), phospho-TAK1 (rabbit, monoclonal), phospho-IKKα/β (rabbit, monoclonal), caspase-3 (rabbit, monoclonal), cleaved caspase-3 (rabbit, monoclonal), Bax (rabbit, monoclonal), Bcl-2 (rabbit, monoclonal), and GAPDH were all provided by Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody was also ordered from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor® 488 dye was provided by Invitrogen.

Yu X., Meng X., Xu M., Zhang X., Zhang Y., Ding G., Huang S., Zhang A, & Jia Z. (2018). Celastrol ameliorates cisplatin nephrotoxicity by inhibiting NF-κB and improving mitochondrial function. EBioMedicine, 36, 266-280.

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Western Blot Analysis of Myocardial Protein Expression

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Myocardial tissue was homogenized with RIPA lysis buffer (Santa Cruz Biotechnology) and protein concentration of the supernatants was determined by BCA Assays (Pierce Biotechnology). Equal amounts of protein (50 μg) were electrophoresed on a 14% SDS-polyacrylamide gel and then electrophoretic-ally transferred on to a PVDF membrane (Millipore). After blocking with 5% non-fat dried skimmed milk in TBS (Tris-buffered saline) containing 0.05% Tween 20 at room temperature (25°C) for 1 h, the membranes were incubated at 4°C with primary antibodies at 1:1000 dilutions on a rocking platform overnight. Antibodies used were phospho-Smad2, Smad2, phospho-TAK1 (TGF-β-activated kinase 1), TAK1, phospho-p38 MAPK (mitogen-activated protein kinase), p38 MAPK, phospho-ERK (extracellular-signal-regulated kinase) 1/2, ERK1/2, phospho-JNK (c-Jun N-terminal kinase), JNK (all from Cell Signaling Technology), TGF-β1 and β-actin (Santa Cruz Biotechnology). Blots were incubated with HRP (horseradish peroxidase)-labelled secondary antibodies (anti-mouse IgG or anti-rabbit IgG) for 1 h at room temperature, and signals were detected using Pierce ECL (enhanced chemiluminescence) Western Blotting Substrate. Membrane was exposed to Fuji radiograph film, and signal intensities were analysed with the NIH ImageJ software (version 1.38×).

ZHANG H., Wu J., DONG H., KHAN S.A., CHU M.L, & TSUDA T. (2014). Fibulin-2 deficiency attenuates angiotensin II-induced cardiac hypertrophy by reducing transforming growth factor-β signalling. Clinical science (London, England : 1979), 126(4), 275-288.

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Protein Extraction and Immunoblotting

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Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM each MgCl₂, MnCl₂ and CaCl₂, 1 mM PMSF and 10 mM sodium fluoride) [23 (link),24 (link)]. Tissues were homogenized in T-PER tissue protein extraction reagent (Thermo, 78510), in which protease inhibitor cocktail (Biotool, B14001) was added. Insoluble material was removed by centrifugation at 10,000 g for 10 min. Proteins were separated by SDS-PAGE under reducing condition, followed by blocking in phosphate-buffered saline/Tween-20 containing 1% BSA. The membrane was incubated with antibodies for CTHRC1 (Huabio), TGFBR1 (Cell Signaling, 3712S), TGFBR2 (Cell Signaling, 11888S), TGFBR3 (Cell Signaling, 2519S), Endoglin (Cell Signaling, 4335S), phospho-Smad2 (Cell Signaling, 3108P), Smad2 (Cell Signaling, 5339P), phospho-Smad3 (Cell Signaling, 9520P), Smad3 (Cell Signaling, 9523P), Smad4 (Cell Signaling, 9515P), phospho-TAK1 (Cell Signaling, 4508S), TAK1 (Cell Signaling, 5206S), phospho-p38 (Cell Signaling, 4511S), p38 (Cell Signaling, 8690S), phosphor-JNK (Cell Signaling, 4668S), JNK (Cell Signaling, 9252S), Wnt5a (Cell Signaling, 2392S), and GAPDH (Huabio, M1211-1), followed by incubation of species-specific secondary antibodies. Bound secondary antibodies (LI-COR, 926-32213; 926-68051) were revealed by Odyssey imaging system (LI-COR).

Li J., Wang Y., Ma M., Jiang S., Zhang X., Zhang Y., Yang X., Xu C., Tian G., Li Q., Wang Y., Zhu L., Nie H., Feng M., Xia Q., Gu J., Xu Q, & Zhang Z. (2019). Autocrine CTHRC1 activates hepatic stellate cells and promotes liver fibrosis by activating TGF-β signaling. EBioMedicine, 40, 43-55.

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Osteoclastogenesis Regulation by Signaling Pathways

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The alpha modification of Eagle’s medium (α-MEM), penicillin/streptomycin, and foetal bovine serum (FBS) were purchased from Gibco-BRL (Gaithersburg, MD, USA). The cell counting kit (CCK-8) was obtained from Dojindo Molecular Technology (Kumamoto, Japan). Recombinant mouse M-CSF and mouse RANKL were obtained from R&D (Minneapolis, MN, USA), and LY was purchased from Sigma Aldrich (St Louis, MO, USA). Specific antibodies against ERK, JNK, P38, IκB-α, phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), MEK1/2, MKK6, MKK7, TAK1, phospho-MEK1/2, phospho-MKK6, phospho-MKK7, phospho-TAK1, NFATc1, and β-actin were obtained from Cell Signalling Technology (Cambridge, MA, USA). The TRAP staining kit, and all other reagents, were purchased from Sigma Aldrich unless otherwise stated.

Chen S., Jin G., Huang K.M., Ma J.J., Wang Q., Ma Y., Tang X.Z., Zhou Z.J., Hu Z.J., Wang J.Y., Qin A, & Fan S.W. (2015). Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo. Scientific Reports, 5, 12853.

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Protein Expression Analysis in Skin Tissue

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Primary antibodies reactive with mouse Sca-1, STAT3, TAK1, phospho-TAK1 (Thr184/187), phospho-STAT3 (Tyr705), and β-actin (Cell Signaling Technology, Danvers, MA), murine mast cell protease-1 (mMCPT1; BioLegend, San Diego, CA), filaggrin (Abcam, Cambridge, MA) and vaccinia antigens (Santa Cruz Biotech, Dallas, TX) were used according to manufacturer recommendations. Formalin-fixed, paraffin-embedded skin tissues were sectioned and processed as previously described [4 (link)]. Cell cytosolic fractionation and immunoblot of HEK-001 cell lysates were analyzed as previously described [4 (link)].

He Y., Sultana I., Takeda K, & Reed J.L. (2017). Cutaneous Deficiency of Filaggrin and STAT3 Exacerbates Vaccinia Disease In Vivo. PLoS ONE, 12(1), e0170070.

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AMPK Regulation by Limonin and Compound C

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Limonin (Lim, CAS# 1,180-71-8, HPLC≥95%) was purchased from TCI (Shanghai) Development Co., Ltd., China. Compound C (#B3252) was purchased from ApexBio, United States. Antibody sources are as follows: phospho-AMPKα (#2535, Cell Signaling Technology), AMPKα (#5831, Cell Signaling Technology), phospho-ACC (#3661, Cell Signaling Technology), ACC (#3662, Cell Signaling Technology), phospho-LKB1 (#3055, Cell Signaling Technology), LKB1 (#3050, Cell Signaling Technology), phospho-CAMKK2 (#12818, Cell Signaling Technology), CAMKK2 (#16810, Cell Signaling Technology), PP2A (#2038, Cell Signaling Technology), PP2C (#3549, Cell Signaling Technology), phospho-TAK1 (#4508, Cell Signaling Technology), TAK1 (#5206, Cell Signaling Technology), SREBP1 (ab28481, Abcam), SERBP2 (ab30682, Abcam), β-actin (#3700, Cell Signaling Technology). Commercial kits used in measurement of plasma parameters are as follows: triglyceride (TG), total cholesterol (TC) assay kits were purchased from Dongou Diagnostics Co., Ltd., Zhejiang, China; alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), non-esterified fatty acid (NEFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.

Wang S.W., Lan T., Chen H.F., Sheng H., Xu C.Y., Xu L.F., Zheng F, & Zhang F. (2022). Limonin, an AMPK Activator, Inhibits Hepatic Lipid Accumulation in High Fat Diet Fed Mice. Frontiers in Pharmacology, 13, 833705.

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Western Blot Analysis of Protein Signaling

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After two washes with phosphate buffered saline (PBS), cells were scrapped on ice into lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with 1 mM PMSF and the soluble fraction was isolated after centrifugation (14000 rpm, 10 min, 4°C). Protein was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol, and lysates were resolved on 8% poly-acrylamide gels and transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% milk in Tris buffered saline with 0.1% Tween-20 (TBST) for 1 hr, followed by an overnight incubation at 4°C in primary antibody (1:1000). Membranes were washed with TBST before and after exposure to goat-anti-rabbit HRP secondary antibody (1 hr; Cell Signaling) and protein were visualized using Kodak 1D Image Analysis Software 3.6 and a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Primary antibodies used for western blotting include phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-AKT, AKT, phospho-JNK, phospho-IKK, phospho-NF-κB, phospho-elF-2α, and phospho-TAK1 were obtained from Cell Signaling, anti-GPR120 was obtained from Abcam and anti-β-actin and anti-TNFα were obtained from Sigma.

Wellhauser L, & Belsham D.D. (2014). Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons. Journal of Neuroinflammation, 11, 60.

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Protein Expression Analysis in Left Atrial Appendage

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Left atrial appendage samples were rinsed in cold PBS, and then placed in a 1.5 ml round-bottom tube. Ice-cold RIPA buffer (Beyotime technology), PMSF (0.5ul/ml) and inhibitor of phosphatase was added at 4°C. Total protein was extracted with Protein Extraction Kit (Thermo Scientific, USA) and protein concentration was measured using the BCA Protein Assay Kit (Thermo Scientific, USA), respectively. Protein samples were separated on 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. After blocking with 5% BSA in tris-buffered saline with tween, the membranes were incubated overnight at 4°C with primary antibodies against TGF-β1, CTGF, TRAF6 (Santa Cruz, USA), extracellular signal-regulated kinase1/2 (ERK1/2), phospho-ERK1/2, P38 MAPK, phospho-P38 MAPK, phospho-c-Jun NH(2)-terminal kinase (JNK), TAK1, phospho-TAK1, GAPDH (Cell Signaling Technology, USA), and JNK (Bioworld, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Western blots were performed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, USA) and quantified by scanning densitometry. The ratio of interested protein was normalized to GAPDH and was densitometrically analyzed by Quantity One software (USA).

Zhang D., Chen X., Wang Q., Wu S., Zheng Y, & Liu X. (2017). Role of the MAPKs/TGF-β1/TRAF6 signaling pathway in postoperative atrial fibrillation. PLoS ONE, 12(3), e0173759.

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Immunohistochemical Analysis of Cell Signaling

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Paraffin-embedded tissue was processed for immunohistochemical localization was performed with antibodies against p-AKT, p-TAK, p-histoneH3 (Cell Signaling), C3 (Santa Cruz Biotechnology), and TUNEL (Thermo Fisher Scientific) as previously described before (58 (link), 59 (link)). All of the slides were scanned using Leica SCN400 (Leica Micro System) and analyzed by Tissue IA Optimizer (Leica). The values of positively stained cells were measured in an unbiased manner. C3a concentration of cultured medium and serum was assayed by sandwich ELISA using a human C3a ELISA kit (BD Bioscience) according to the manufacturer’s instructions. Western blots separated by 10, 12, or 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis were incubated with primary antibodies for TLR9, DEC205 (LS Bio Seattle), phospho-TAK1, TAK1, phospho-AKT, AKT, phospho-ERK1/2, ERK, BCL2 (Cell Signaling), and C3 (Santa Cruz Biotechnology). Western blots were visualized using alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich). The ELISA for anaphylatoxin C3a was performed according to manufacturer guidelines (LSBio).

Haldar S., Mishra R., Billet S., Thiruvalluvan M., Placencio-Hickok V.R., Madhav A., Duong F., Angara B., Agarwal P., Tighiouart M., Posadas E.M, & Bhowmick N.A. (2020). Cancer epithelia-derived mitochondrial DNA is a targetable initiator of a paracrine signaling loop that confers taxane resistance. Proceedings of the National Academy of Sciences of the United States of America, 117(15), 8515-8523.

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Western Blot Analysis of Signaling Proteins

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Equal amounts of protein were separated using SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were incubated with antibodies against phospho-eNOS, total-eNOS, phospho-TAK1, total-TAK1, phospho-JNK, total-JNK, phospho-p38 MAPK, total-p38 MAPK, phospho-ERK1/2, total-ERK1/2 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) (Table S2). The relative density was calculated as the ratio of the intensity of the protein of interest to that of β-actin, and all band intensities were within the linear range.

Jung E., Baek E.B., Hong E.J., Kang J.H., Park S., Park S., Hong E.J., Cho Y.E., Ko J.W., Won Y.S, & Kwon H.J. (2024). TXNIP in liver sinusoidal endothelial cells ameliorates alcohol-associated liver disease via nitric oxide production. International Journal of Biological Sciences, 20(2), 606-620.

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