Total erk1 2

Manufactured by Cell Signaling Technology

Sourced in United States, Germany, United Kingdom

Total ERK1/2 is a laboratory assay that quantifies the total levels of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) proteins in a sample. ERK1/2 are important signaling molecules involved in cellular processes such as proliferation, differentiation, and survival.

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ERK1/2 (1487 citations) Anti-total ERK1/2 (50 citations) Rabbit anti-total-ERK1/2 (10 citations) Anti-total ERK1/2 antibody (7 citations) Total p44/42 MAPK (ERK1/2) (6 citations) Total p44/42 ERK1/2 (5 citations) Phospho- and total-Erk1/2 (4 citations) Antibodies against phospho-ERK1/2 and total ERK1/2 (4 citations) Total ERK1/2 (t-ERK1/2, (4 citations) Anti total and phospho-Erk1/2 (4 citations)

239 protocols using total erk1 2

Neurochemical Profile of Neuroinflammation

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Phospho-Erk (p44/42 mitogen activated protein kinase), Total Erk 1/2, and phospho-CEBP-β from Cell Signaling (Danvers, MA), Peripherin (Millipore, MA), choline acetyl transferase (Millipore, MA), SERT (Abcam), Hand-2 (Invitrogen), N-acetyl cysteine and fMLF (Sigma), ROSstar™ 550 (Hydrocyanine-3 probe) from LI-COR ^{20 (link), 21 (link)}.

Chandrasekharan B., Saeedi B.J., Alam A., Houser M., Srinivasan S., Tansey M., Jones R., Nusrat A, & Neish A.S. (2019). Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice. Gastroenterology, 157(1), 179-192.e2.

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Ni(SalPipNONO) Mechanism in A549 Cells

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Sub-confluent A549 were seeded in 6 cm diameter Petri dishes. After adherence, cells were treated for the indicated times with Ni(SalPipNONO) (0.5 mM) and specific pathway inhibitors. Protein extraction and Western blot were performed as previously described [58 (link), 61 (link)]. Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [Anti-cytochrome c, anti-phospho-ERK1/2, anti-caspase- 3, anti-ERK antibodies from Cell Signaling (Celbio, Milan, Italy); anti-p53 antibody from Santa Cruz Biotechnology Inc (Dallas TX, USA); anti-COX-2 from Cayman Chemical (Ann Arbor, MI, USA); anti-HIF-1α from BD Biosciences (San Jose, CA, USA); anti-VEGF from Merck KGaA (Darmstadt, Germany)] and then detected by enhanced chemiluminescence system (Biorad, Hercules, CA, USA). Results were normalized to those obtained by using an antibody against beta actin from Merck KGaA (Darmstadt, Germany) or total ERK1/2 (Cell Signaling, Celbio, Milan, Italy), when indicated.

Ciccone V., Monti M., Monzani E., Casella L, & Morbidelli L. (2018). The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis. Oncotarget, 9(17), 13353-13365.

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Western Blot Analysis of Ion Channels

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The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20–40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for Ca_V3.1 and Ca_V3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: Ca_V3.1 (1:1000) (Alomone, Jerusalem, Israel) and Ca_V3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the blots were washed thrice with 1 × TBS + 0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1 × TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.

Pandi S.P., Shattock M.J., Hendry B.M, & Sharpe C.C. (2022). Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1. BMC Nephrology, 23, 211.

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Western Blot Analysis of Cell Signaling Proteins

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Western blot analysis was performed essentially as described [19 (link)]. The method used to normalize the protein levels was “Pierce BCA Protein Assay Kit” (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (15 μg total protein per lane) were prepared in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-PAGE, and blotted on polyvinylidene difluoride membranes. The following antibodies were used: mouse anti-human β-actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), phosphor-P70S6K (Cell Signaling, MA, USA), total P70S6K (Cell Signaling, MA, USA), pERK1/2 (Cell Signaling, MA, USA), total ERK1/2 (Cell Signaling, MA, USA), anti-mouse IgG HRP conjugate (Proteintech, IL, USA), and rabbit anti-rat IgG HRP (Proteintech, IL, USA). Image J software was used for densitometric analyses.

Chinnapaka S., Yang K.S., Flowers Q., Faisal M., Nerone W.V., Rubin J.P, & Ejaz A. (2021). Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling. Biomedicines, 9(12), 1782.

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Western Blot Analysis of Protein Signaling

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Human ORS and DP cells or skin tissue were lysed (RIPA lysis buffer; #20‐188; Merck Millipore). The obtained protein was then separated by 8% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Amersham). The protein transferred membranes were incubated overnight with the specific antibodies. The following antibodies were used: β‐actin (#MA5‐15739, 1:5,000; Thermo Fisher), phospho‐ERK1/2 (#9101, 1:1,000; Cell Signaling), total ERK1/2 (#9102, 1:1,000; Cell Signaling), phospho‐AMPK (#2535, 1:1,000; Cell Signaling), and total AMPK (#2532, 1:1,000; Cell Signaling). Then, the membranes were washed and incubated with anti‐rabbit IgG or anti‐mouse IgG antibodies (horseradish peroxidase‐conjugated, GTX213110, GTX213111, 1:10,000; GeneTex) at 25°C for 1 h. Antibody‐antigen complexes detected by enhanced chemiluminescent substrate (Thermo Fisher) were captured and quantified by Amersham imager 680 systems (GE Healthcare).

Ohn J., Been K.W., Kim J.Y., Kim E.J., Park T., Yoon H., Ji J.S., Okada‐Iwabu M., Iwabu M., Yamauchi T., Kim Y.K., Seok C., Kwon O., Kim K.H., Lee H.H, & Chung J.H. (2021). Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth. EMBO Molecular Medicine, 13(10), e13790.

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ERK1/2 Activation in HT-1080 Cells

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200,000 HT-1080 cells/well were grown overnight in a 6-well tissue culture plate. Cells were washed with 1 mL HBSS and the media was replaced with serum-free DMEM High Glucose media containing 1% Pen/Strep. After 16 h serum starvation, HT-1080 cells were stimulated with serum-containing media ± U0126 (5 μM) or trametinib (250 nM) for 30 min, washed with 1 mL HBSS and lysed in 100 μL of 9 M urea containing protease and phosphatase inhibitor cocktail (Cell Signaling Technology). Samples were analyzed by Western blotting as described above. Rabbit monoclonal antibodies directed against the diphosphorylated (Thr²⁰²/Tyr²⁰⁴) form of ERK1/2 (p42/44 MAPK, Cell Signaling Technology, dilution: 1:1000) and total ERK1/2 (Cell Signaling Technology, dilution: 1:1000) were the primary antibodies.

Conlon M., Poltorack C.D., Forcina G.C., Armenta D.A., Mallais M., Perez M.A., Wells A., Kahanu A., Magtanong L., Watts J.L., Pratt D.A, & Dixon S.J. (2021). A Compendium of Kinetic Modulatory Profiles Identifies Ferroptosis Regulators. Nature chemical biology, 17(6), 665-674.

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Insulin and IGF-I Signaling Pathway Analysis

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Following confluency, pOBs were cultured for 24 hours in serum-free medium containing 0.1% BSA. Cells were then either lyzed immediately or stimulated with insulin (10 nM; Sigma) or IGF-I (10 nM; Sigma) for 10 minutes before lysis. Cells were lysed in RIPA buffer (Invitrogen) containing ‘phosphatase inhibitor cocktail 2’ (Sigma) and ‘complete’ protease inhibitor cocktail (Roche) according to the manufacturers’ instructions. Immunoblotting was conducted with specific antibodies against phospho-Akt^Ser473, total Akt, phospho-GSK3β^Ser9, total GSK3β, phospho-Erk1/2^{Thr202/Tyr204} and total Erk1/2 (Cell Signaling, Boston, MA, USA). Immunoblotting was conducted as previously described (Zhu et al., 2011 (link); Zhu et al., 2013 (link)) and visualized using the enhanced chemiluminescence (ECL) Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK).

Huesa C., Zhu D., Glover J.D., Ferron M., Karsenty G., Milne E.M., Millan J.L., Ahmed S.F., Farquharson C., Morton N.M, & MacRae V.E. (2014). Deficiency of the bone mineralization inhibitor NPP1 protects mice against obesity and diabetes. Disease Models & Mechanisms, 7(12), 1341-1350.

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Western Blot Analysis of EZH2, AKT, and ERK

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Tissues were lysed in 1× lysis buffer (Cell Signaling Technology, #9803) with protease inhibitor cocktail (EMD Biosciences, 539131). Cell lysates were separated using standard western blotting procedures, and membranes were probed with the following antibodies: mouse monoclonal EZH2 (BD Transduction Laboratories, #612666) at 1:1000; total AKT (Cell Signaling Technology, #9272) at 1:2000; total Erk1,2 (Cell Signaling Technology, #4695S) at 1:2000; pAKT antibody (Cell Signaling Technology, #4060S) at 1:1000; pERK1,2 (Cell Signaling Technology, #4376S) at 1:1000) all incubated overnight at 4µC. β-actin-HRP (SIGMA A3854, 1:20000) was used as loading control. Secondary antibody Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling 7074, 1:2000) or Anti-mouse IgG, HRP-linked Antibody (Cell Signaling 7076, 1:2000) was incubated for 1 hour at room temperature. After washing, chemiluminescence was visualized with Western Lightning Plus-ECL (PerkinElmer) and exposure onto KODAK BioMax XAR film. Relative quantifications were performed using ImageJ software.

Zhang H., Qi J., Reyes J.M., Li L., Rao P.K., Li F., Lin C.Y., Perry J.A., Lawlor M.A., Federation A., De Raedt T., Li Y.Y., Liu Y., Duarte M.A., Zhang Y., Herter-Sprie G.S., Kikuchi E., Carretero J., Perou C.M., Reibel J.B., Paulk J., Bronson R.T., Watanabe H., Fillmore C.M., Kim C.F., Hammerman P.S., Brown M., Cichowski K., Long H., Bradner J.E, & Wong K.K. (2016). Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer. Cancer discovery, 6(9), 1006-1021.

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Quantifying Renal Protein Expression

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The remaining renal tissue was snap frozen in liquid nitrogen. Kidney samples were homogenised in RIPA buffer for protein analysis by western blotting. Protein concentration was measured before electrophoresing on 12% (w/v) SDS- polyacrylamide gels. After electrotransfer to Immobilon-P membranes (Millipore, USA), membranes were blocked at room temperature with 10% (w/v) milk powder for 1 hour. The primary antibodies against pERK1/2 (1:1000, Cell Signalling, USA), total ERK1/2 (1:1000, Cell Signalling), calnexin (1:1000, Sigma Aldrich, UK), phospho-cJun(p-cjun) (1:1000, Cell Signalling), phospho-p38(p-p38) (1:1000, Cell Signalling) and PAI-1 (1:1000, Abcam, UK) were incubated overnight at 4°C. After washing, the membranes were incubated with goat IgG-horseradish peroxidise conjugated secondary antibodies (DAKO) at a final dilution of 1:20,000 for 1 hour at room temperature. After washing, antibody binding was visualised with enhanced chemiluminescence (Roche, UK) and autoradiography. The expression levels of proteins were analysed on western blots by using 20μg of total cell lysates with loading errors corrected for using calnexin due to its different molecular weight.

Nutter F.H., Haylor J.L, & Khwaja A. (2015). Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis. PLoS ONE, 10(9), e0137321.

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Protein Extraction and Western Blot Analysis Protocol

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For western blot analysis, snap-frozen tissues were homogenized on ice in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 10% glycerol, 1% NP-40, 0.5% Triton X-100 and protease inhibitors. Samples were centrifuged for 5 min at 0.5 × g at 4°C and infranatant collected. An appropriate volume of 6X Laemmli buffer was added and all samples incubated at 100°C for 10 minutes and then briefly placed on ice. Protein concentration was determined by Bradford assay and then equal quantity of sample resolved by SDS-PAGE. Proteins were transferred to a PVDF membrane before being probed with the specified antibodies. Antibody information: β-actin (Sigma, #A5316), phospho-ERK1/2 (Cell Signaling, #9101), total ERK1/2 (Cell Signaling, #9102), Myc (Millipore, #05–724) and β-klotho (R&D Systems, #AF2619).

BonDurant L.D., Ameka M., Naber M.C., Markan K.R., Idiga S.O., Acevedo M.R., Walsh S.A., Ornitz D.M, & Potthoff M.J. (2017). FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms. Cell metabolism, 25(4), 935-944.e4.

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