T erk

Manufactured by Cell Signaling Technology

Sourced in United States

T-ERK is a laboratory equipment product developed by Cell Signaling Technology. It is used to detect and quantify the total levels of extracellular signal-regulated kinase (ERK) proteins in cell and tissue samples.

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Lab products found in correlation

59 protocols using t erk

Comprehensive Signaling Pathway Analysis in NOTCH1-ROS1 Expressing Cells

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NOTCH1–ROS1 expressing NIH3T3 and HEK293 cells were lysed in RIPA buffer and normalized using a Pierce 660-nm Protein Assay. Lysates were run on NuPAGE precast gels (4%–12% Bis-Tris or 3%–8% Tris-Acetate; Invitrogen). For MAPK, PI3K, and JAK/STAT pathway analysis, pMEK (#9154), tMEK (#4694), pERK (#4370), pERK (#9101), tERK (#4695), tERK (#4696), pAKT Thr308 (#4056), pAKT Ser473 (#9271), pAKT Ser473 (#4060), tAKT (#2920), pS6 (#4858), tS6(#2317), pSTAT3 (#9145), tSTAT3 (#9139), pROS1 Tyr2274 (#3078), tROS1 (#15027), and GAPDH (#3683) antibodies were purchased from Cell Signaling (1:1000).

Jain P., Iyer S., Straka J., Surrey L.F., Pogoriler J., Han H., Smith T., Busch C., Fox E., Li M., Waanders A.J., Resnick A, & Davare M.A. (2022). Discovery and functional characterization of the oncogenicity and targetability of a novel NOTCH1–ROS1 gene fusion in pediatric angiosarcoma. Cold Spring Harbor Molecular Case Studies, 8(6), a006222.

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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).

Smith A.G., Kliebe V.M., Mishra S., McCall R.P., Irvine M.M., Blagg B.S, & Lei W. (2024). Anti-inflammatory activities of novel heat shock protein 90 isoform selective inhibitors in BV-2 microglial cells. Frontiers in Molecular Biosciences, 11, 1405339.

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Molecular Signaling Pathways Regulation

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Piperine (≥ 97% purity, as determined by high-performance liquid chromatography) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Isoprenaline (ISO, I5627), TGF-β (T7039), angiotensin II (Ang II, A9525) and phenylephrine (PE, P6126) were purchased from Sigma-Aldrich. The primary antibodies against the following proteins were purchased from Cell Signaling Technology: T-AKT (4691, 1:1000), phospho-AKT (P-AKT, 4060, 1:1000), T-glycogen synthase kinase 3β (GSK3β, 9315, 1:1000), P-GSK3β (9323P, 1:1000), T-P38 (9212P, 1:1000), P-P38 (4511P, 1:1000), T-AMPKα (2603P, 1:1000), P-AMPKα (2535, 1:1000), T-ERK (4695, 1:1000), P-ERK (4370P, 1:1000), T-SMAD3 (3103S, 1:500), P-SMAD3 (3101, 1:500) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 2118, 1:1000). α-Smooth muscle actin (α-SMA, ab7817, 1:500) was obtained from Abcam (Cambridge, UK). Antibodies against α-actinin (ab90776) were obtained from Merck Millipore (Massachusetts, United States), and anti-vimentin (sc-5565) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-rabbit/mouse EnVisionTM +/HRP reagent used for immunohistochemistry was purchased from Gene Technology (Shanghai, China), and Alexa Fluor 488-goat anti-mouse secondary antibody was purchased from LI-COR Biosciences (Lincoln, USA). The BCA protein assay kit was from Pierce (Rockford, IL, USA).

Ma Z.G., Yuan Y.P., Zhang X., Xu S.C., Wang S.S, & Tang Q.Z. (2017). Piperine Attenuates Pathological Cardiac Fibrosis Via PPAR-γ/AKT Pathways. EBioMedicine, 18, 179-187.

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Western Blot Analysis of Neuronal Proteins

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Cell and brain samples were lysed using a sonicator (Qsonica), and were subjected to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad). Separated proteins were transferred onto PVDF membranes (Bio-Rad) for Western blotting, and then hybridized with the primary antibodies, including P-ERK (Cell Signaling; 1:1,000 dilution), T-ERK (Cell Signaling; 1:2,000 dilution), P-GAP43 (Abcam; 1:1,000 dilution), T-GAP43 (Genetex; 1:5,000 dilution), PSD95 (Abcam; 1:5,000 dilution), Synaptophysin (Abcam; 1:5,000 dilution), VAMP1 (Abcam; 1:5,000 dilution), RANBP10 (Proteintech; 1:2,000), Flag M5 (Sigma; 1:2,000 dilution), mEM48 (a gift from Dr. Xiao-Jiang Li, Emory University, USA; 1:50 dilution), MAB2166 (Millipore; 1:2,000 dilution) and γ-tubulin (Sigma; 1:10,000 dilution) antibodies. Protein expression levels were determined by an Amersham ECL kit (PerkinElmer) through an imaging system (ChampGel), and expression profiling was quantitated by an ImageJ system (NIH).

Her L.S., Mao S.H., Chang C.Y., Cheng P.H., Chang Y.F., Yang H.I., Chen C.M, & Yang S.H. (2017). miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease. Theranostics, 7(9), 2452-2462.

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Protein Expression Analysis of UCB-MSC Spheroids

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Proteins were collected from 3D spheroids of UCB-MSCs using the radioimmunoprecipitation assay buffer (RIPA), which is a lysis buffer containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA); proteins were then sonicated. Protein samples were separated and transferred onto nitrocellulose membranes. After blocking, the membranes were stained with primary antibodies: PARP, p-ERK, t-ERK, p-AKT and t-AKT (cell signaling, Danvers, MA, USA), BAX, SOD2, E-cadherin, PI3K and p-Nrf2 (Abcam, Cambridge, UK), caspase-3 (Santa Cruz Biotech., Dallas, TX, USA), GAPDH (AbClon, Seoul, Korea), and β-actin (Sigma), respectively. After washing, the membranes were incubated with the HRP-conjugated secondary antibody. Protein bands were visualized using an Amersham™ ECL Plus system (GE Healthcare, Chicago, IL, USA) and imaged using a ChemiDoc XRS camera (Bio-Rad Laboratories, Hercules, CA, USA). Band densities were analyzed via Image Lab software 6.0.1 (BioRad) and calculated by normalization to GAPDH or β-actin.

Lee M., Song B.R., Kim D.H., Ha J., Lee M., Choi S.J., Oh W., Um S, & Jin H.J. (2020). Up-Regulation of Superoxide Dismutase 2 in 3D Spheroid Formation Promotes Therapeutic Potency of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells. Antioxidants, 9(1), 66.

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Western Blot and Cell Fractionation Analysis

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Western blot analysis and cell fractionation were carried out as described previously [32 (link)]. Labelled bands were detected by Immun-Star horseradish peroxidase chemiluminescence kit (Bio-Rad, Regents Park, NSW, Australia), and images were captured with the Fujifilm LAS-4000 image system (Fujifilm, Brookvale, NSW, Australia). Antibodies included in this study were against PD-L1 (Rand D, Minneapolis, MN, USA) p-ERK, t-ERK, cyclin D1, STAT3, p50/105, RelA/p65 (D14E15), actin, tubulin, PARP (Cell Signaling, Danvers, MA, USA); XIAP (20/hILP/X, BD) GAPDH, c-Jun, p-c-Jun and vinculin (Santa Cruz technologies, Dallas, TX, USA).

Gowrishankar K., Gunatilake D., Gallagher S.J., Tiffen J., Rizos H, & Hersey P. (2015). Inducible but Not Constitutive Expression of PD-L1 in Human Melanoma Cells Is Dependent on Activation of NF-κB. PLoS ONE, 10(4), e0123410.

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Protein Expression Analysis in Heart Tissues

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Total proteins isolated from the heart tissues were size fractionated using SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Billerica, MA). The blotted membranes were incubated with antibodies against p-ERK, t-ERK, p-ErbB2, p-ErbB4, LC3b (Cell Signaling Technology, Beverly, MA), β₁-AR (beta-1 adrenergic receptor; Abcam, Cambridge, MA), C/EBPβ, and AT₁-R, (Santa Cruz Biotechnology Inc, Santa Cruz, CA) and with an HRP-conjugated secondary antibody (1:5000, KangChen Biotechnology, Shanghai, China). Either GAPDH or t-ERK was used as an internal control. The proteins were visualized using an ECL Western blotting detection system (GE Healthcare, catalog number RPN2106). The relative intensities of the protein bands were analyzed through densitometry with a gel documentation system using LAS-300 image analysis software. All experiments were repeated at least 3 times.

Ye Y., Gong H., Wang X., Wu J., Wang S., Yuan J., Yin P., Jiang G., Li Y., Ding Z., Zhang W., Zhou J., Ge J, & Zou Y. (2015). Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload–induced Cardiac Hypertrophy and Remodeling in Male Mice. Journal of Cardiovascular Pharmacology, 65(6), 628-639.

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Angiotensin II-Induced Signaling Pathway

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Ang II was purchased from ENZO (ALX-151-039-M025); collagenase and trypsin were purchased from Gibco (Grand Island, NY, United States); the BCA protein assay kit was bought from Pierce (Rockford, United States); and 2,7-dichlorofluorescindiacetate (DCFH-DA) was obtained from the Bioengineering Institute (Nanjing, China). The following primary antibodies were obtained from Cell Signaling Technology (CST, United States): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118), p-mTORC1 (#2971), T-mTORC1 (#2983), α-actinin (#69758S), P-p70 S6 kinase (Thr389) (#9234P), T-p70 S6 kinase (#2708), P-JNK (T183/Y185) (#4668P),T-JNK (#9258), P-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370P), T-ERK (#4695), P-p38 (#4511P), p38 MAPK (#9212P), T-TAK1 (#5206), P-TAK1 (#4508), T-AKT (#4691), P-AKT (#4060), acetyl-CoA carboxylase antibody (#3676), and P-acetyl-CoA carboxylase antibody (#3661S). ABCAM provided the following primary antibodies: Anti-AMPKα2 (ab3760), p-AMPKα2 (S491, ab109402), anti-SOD1 (ab16831), anti-SOD2 (ab38155), Nrf2 (ab15323), 4-hydroxynonenal (ab46545), sarcomeric α-actinin (ab68167), heme oxygenase1 (ab-13243), and NOX2/gp91phox (ab129068). The Sesn2 antibody was acquired from Proteintech (no. 10795-1-AP). Antibodies were used at 1:1,000 dilutions for Western blotting. The secondary antibodies were obtained from LI-COR Biosciences (Lincoln, United States).

Zhang N., Liao H.H., Feng H., Mou S.Q., Li W.J., Aiyasiding X., Lin Z., Ding W., Zhou Z.Y., Yan H., Chen S, & Tang Q.Z. (2021). Knockout of AMPKα2 Blocked the Protection of Sestrin2 Overexpression Against Cardiac Hypertrophy Induced by Pressure Overload. Frontiers in Pharmacology, 12, 716884.

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Protein Extraction and Western Blotting

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Leukemic cells lines were harvested by centrifugation at 300xg for 5 minutes, lysed in buffer containing 20 mM TrisHCl pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% NP-40, 1 mM Na3VO4, 50 mM NaF, 2 mM PMSF and protease inhibitor cocktail (Roche). Transduced, sorted BM cell were lysed in 1X Laemmli buffer supplemented with 1 mM Na3VO4, 50 mM NaF, 2 mM PMSF and protease inhibitor cocktail. The total protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific). 5-20 μg of protein were analyzed on 10% SDS-PAGE gels. The proteins were transferred to ImmobilonP PVDF membranes (Milipore) activated in ethanol, by overnight wet blotting in Tris-Glycine. The membranes were blocked for 1hr in 5% milk in TBS-T, and incubated for 1hr at room temperature or overnight at 4°C with primary antibodies (MEK1 (Cell Signaling #2352), MEK2 (BD Transduction Laboratories #610236), GFP (Roche #11814460001), GAPDH (Milipore #abs16), TUBA (Sigma #T9026), tERK (Cell Signaling #9102), pERK(Cell Signaling #9101), CMYC (Abcam #ab32072) and NRAS (Santa Cruz #sc-31) and developed using SuperSignal West Pico/Femto Chemiluminescent Substrate (Thermo Scientific), Amersham Hyperfilm (Ge Healthcare) and CURIX 60 processor (Agfa) or ChemiDoc Touch Imaging System (Bio-Rad). Densitometric quantification of immunoblots was performed using ImageLab software.

Nowacka J.D., Baumgartner C., Pelorosso C., Roth M., Zuber J, & Baccarini M. (2016). MEK1 is required for the development of NRAS-driven leukemia. Oncotarget, 7(49), 80113-80130.

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Immunoblotting for Cell Signaling

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All chemicals were obtained from Sigma-Aldrich and antibodies were purchased from BioLegend if not stated otherwise. Antibodies for p-ZAP70 (1:500), p-ERK (1:1,000) and t-ERK (1:2,000) were from Cell Signaling and anti-EB1 (1:500) from Absea.

Herter J.M., Grabie N., Cullere X., Azcutia V., Rosetti F., Bennett P., Herter-Sprie G.S., Elyaman W., Luscinskas F.W., Lichtman A.H, & Mayadas T.N. (2015). AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation. Nature Communications, 6, 10182.

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