Total erk

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Germany

Total ERK is a lab equipment product that measures the total amount of extracellular signal-regulated kinase (ERK) protein in a sample. ERK is a key signaling molecule involved in various cellular processes. This product provides a quantitative assay to determine the overall levels of ERK protein in a given biological sample.

Automatically generated - may contain errors

Lab products found in correlation

184 protocols using total erk

Western Blotting Analysis of Protein Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein lysates (20 µg) were resolved on a 4–12% bis-tris gel and transferred to Immobilon PVDF transfer membranes. Membranes were incubated for 40 min at room temperature in blocking solution (TRIS-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20), followed by an overnight incubation in primary antibodies at 4°C. Membranes were then washed 3 times and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h. After 3 additional washes, the immune complexes on the membranes were visualized by ECL detection.
The following antibodies were purchased and utilized in our study: Cell Signaling (Danvers, MA): phospho-AKT (#4058), total AKT (#2920), AKT2 (#3063), phospho-ERK 1/2 (#4695), total ERK ½ (#9107), PTEN (#9559), phospho-mTOR (#2971), total mTOR (#2972), phospho-MET (#3129 for western blotting and #3077 for IHC), c-MET (#3148), CD44 (#3570), phospho-Beta catenin (#4176), phospho-FAK (#8556), and total FAK (#3285). Santa Cruz (Dallas, TX): AKT 1 (#5298). BD (San Jose, California): Beta catenin (#610154) and E-cadherin (#610404). Abcam (Cambridge, MA): KRAS (#55391). Millipore (Billerica, MA): p85α (#05-212). All antibodies were used at a concentration of 1∶1,000.

Elliott V.A., Rychahou P., Zaytseva Y.Y, & Evers B.M. (2014). Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model. PLoS ONE, 9(5), e97432.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Levels

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein extracts were separated on 4–12% Tris-glycine gels and electro-blotted onto PVDF membranes as previously described (10 (link)). Protein levels were assessed using antibodies against c-Myc (cat #9405, Cell Signaling, Danvers, MA 01923), p-Myc (cat #13748, Cell Signaling, Danvers, MA 01923), p-ERK½ (cat #4370, Cell Signaling, Danvers, MA 01923), total ERK½ (cat #9102, Cell Signaling, Danvers, MA 01923), GAPDH (cat #5174, Cell Signaling, Danvers, MA 01923), and β-actin (cat #3700, Cell Signaling, Danvers, MA 01923). Densitometry was performed using ImageJ (NIH, Bethesda, MD) as previously described (10 (link)).

Parasido E., Avetian G., Naeem A., Graham G., Pishvaian M., Glasgow E., Mudambi S., Lee Y., Ihemelandu C., Choudhry M., Peran I., Banerjee P.P., Avantaggiati M.L., Bryant K., Baldelli E., Pierobon M., Liotta L., Petricoin E., Fricke S.T., Sebastian A., Cozzitorto J., Loots G.G., Kumar D., Byers S., Londin E., Difeo A., Narla G., Winter J., Brody J.R., Rodriguez O, & Albanese C. (2019). The sustained induction of c-Myc drives nab-paclitaxel resistance in primary pancreatic ductal carcinoma cells.. Molecular cancer research : MCR, 17(9), 1815-1827.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blot analysis was performed using standard protocols. Whole cell lysates were prepared using RIPA lysis buffer (Thermo Scientific). Proteins were separated by SDS -PAGE, followed by standard immunoblot analysis using phosphor-Erk ½, total Erk ½, PTEN (all from Cell Signaling Technology), GAPDH (Millipore), V5 (Life Technologies) and β-actin (Santa Cruz Biotechnology). The expression of gp100 was detected using α-PEP13 antisera (gift from Dr. Vincent Hearing, National Cancer Institute, NIH, Bethesda, MD).

Acquavella N., Clever D., Yu Z., Roelke-Parker M., Palmer D.C., Xi L., Pflicke H., Ji Y., Gros A., Hanada K.I., Goldlust I.S., Mehta G.U., Klebanoff C.A., Crompton J.G., Sukumar M., Morrow J.J., Franco Z., Gattinoni L., Liu H., Wang E., Marincola F., Stroncek D.F., Lee C.C., Raffeld M., Bosenberg M.W., Roychoudhuri R, & Restifo N.P. (2014). Type-1-cytokines synergize with oncogene inhibition to induce tumor growth arrest. Cancer immunology research, 3(1), 37-47.

+ Open protocol

+ Expand

Antibody Staining and Western Blotting for Protein Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibody stains were performed as previously described^{51 (link)} and the pigment on fish was bleached after the antigen retrieval step with a 1% H₂O₂/3% KOH solution. Antibodies used were total ERK (1:500, Cell Signaling 4696) and phospho-histone H3 (1/500, Millipore 06-570).
For western blotting, whole adult brains were dissected and homogenized in RIPA buffer with protease inhibitors. Anti-α-tubulin from Sigma (T6074-200UL (020M4753)) was used as the control at 1/40,000 for approximately 45 min at room temperature, followed by horseradish-peroxidase-coupled mouse secondary antibody for 1 h at room temperature. RhoA antibody from Cytoskeleton (http://www.cytoskeleton.com/arh04) was used at 1/500 overnight at 4 °C. Immunohistochemistry was performed as in ref. 51 (link) with RhoA antibody from Cytoskeleton (http://www.cytoskeleton.com/arh04) freshly diluted in PBS at 1/100.

Escamilla C.O., Filonova I., Walker A.K., Xuan Z.X., Holehonnur R., Espinosa F., Liu S., Thyme S.B., López-García I.A., Mendoza D.B., Usui N., Ellegood J., Eisch A.J., Konopka G., Lerch J.P., Schier A.F., Speed H.E, & Powell C.M. (2017). Kctd13 deletion reduces synaptic transmission via increased RhoA. Nature, 551(7679), 227-231.

+ Open protocol

+ Expand

Protein Isolation and Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein isolation was performed using frozen liver tissue in radioimmunoprecipitation assay buffer with proteinase and phosphatase inhibitors. Pooled protein lysates were utilized for Western blot analysis. Antibodies utilized for Western blot analysis include the following: from Cell Signaling Technologies: cyclin D1 (number 2978), phosphorylated ERK (p44/42; number 9101), total ERK (number 4696), phosphorylated YAP (S127; number 4911), phosphorylated YAP (S397; number 13619), total YAP (number 14074), p16 (number 80772), p27 (number 3686), glyceraldehyde-3-phosphate dehydrogenase (number 5174), phosphorylated epidermal growth factor receptor (EGFR) 846 (number 2231), phosphorylated EGFR 1068 (number 3777), total EGFR (number 4267), phosphorylated Met 1349 (number 3121), and total Met (number 8191). The antibody used for p21 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX; sc6246). Total CAR (number PA5-95066) was purchased from Thermo Fisher Scientific (Waltham, MA).

Koral K., Bhushan B., Orr A., Stoops J., Bowen W.C., Copeland M.A., Locker J., Mars W.M, & Michalopoulos G.K. (2022). Lymphocyte-Specific Protein-1 Suppresses Xenobiotic-Induced Constitutive Androstane Receptor and Subsequent Yes-Associated Protein–Activated Hepatocyte Proliferation. The American Journal of Pathology, 192(6), 887-903.

+ Open protocol

+ Expand

Investigating Oxidative Stress Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary antibodies against gp91phox/NOX2, p47phox, acetylated α-tubulin, p65, IkB, c-fos, and c-jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Total-ERK, total-p38, total-JNK, phosphor-ERK, phosphor-p38, phosphor-JNK, β-actin, and HDAC6 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Hindsiipropane B was synthesized from commercially available starting materials with the Wittig-Horner reaction, Claisen-Schmidt condensation and hydrogenation as key steps (14 (link)). 10 mM stock solution of hindsiipropane B was prepared in dimethyl sulfoxide. Oligonucleotide primers (HDAC6, CCL2, CXCL8, CXCL10, Nox2, p22phox, p47phox, and β-actin) (Bioneer, Seoul, Korea) were obtained from commercially.

Jo H., Jang H.Y., Youn G.S., Kim D., Lee C.Y., Jang J.H., Choi S.Y., Jun J.G, & Park J. (2018). Hindsiipropane B alleviates HIV-1 Tat-induced inflammatory responses by suppressing HDAC6-NADPH oxidase-ROS axis in astrocytes. BMB Reports, 51(8), 394-399.

+ Open protocol

+ Expand

Detailed Molecular Signaling Pathway Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

FGF9 (catalogue # 273-F9-025) was purchased from R&D Systems. METAFECTENE® PRO (#T040) was purchased from Biontex. TRPA1 antibodies were purchased from Alomone (#ACC-037) and Santa-Cruz (#sc-166469). Total FGFR2 antibody (#sc-122) was purchased from Santa-Cruz. pFGFR2 (#3471), pERK (#4370), Total ERK (#9102) β-actin (#4970), Flag (#8146), Ki-67 (#9449), β-tubulin (#2146), Zona Occludin-1 (#5406), Claudin-5 (#4933), IgG antibody (#2729), anti-GFP (#2956) antibodies along with U0126 (#9903) were purchased from Cell Signalling. U73122 (#1268) was purchased from TOCRIS. NEB 5-alpha competent E.Coli (high efficiency) (#C2987H) cells purchased from New England Biolabs, were used for transformation. TRPA1 inhibitor HC030031 (#2896) was purchased from Tocris bioscience. ATP (#9804) and kinase buffer (#9802) were purchased from Cell Signalling. Occludin (H-279) (sc-5562) antibody, validated shRNAs^{18 (link), 55 (link)} (sc-29218-V, sc-108080, and sc-29452-V), and DMA (sc-202459) were purchased from Santa Cruz Biotechnology, Inc. Strep antibody (# ab76949) was bought from abcam. CD-63 antibody (#GTX41877) from GeneTex was utilized as an exosomal marker (exosomal proteins were extracted using the Total Exosome RNA and Protein Isolation kit (#4478545, Invitrogen). All antibodies were used in 1:1000 dilutions for western blots.

Berrout J., Kyriakopoulou E., Moparthi L., Hogea A.S., Berrout L., Ivan C., Lorger M., Boyle J., Peers C., Muench S., Gomez J.E., Hu X., Hurst C., Hall T., Umamaheswaran S., Wesley L., Gagea M., Shires M., Manfield I., Knowles M.A., Davies S., Suhling K., Gonzalez Y.T., Carragher N., Macleod K., Abbott N.J., Calin G.A., Gamper N., Zygmunt P.M, & Timsah Z. (2017). TRPA1–FGFR2 binding event is a regulatory oncogenic driver modulated by miRNA-142-3p. Nature Communications, 8, 947.

+ Open protocol

+ Expand

Protein Extraction and Immunoblotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

Proteins extraction and blotting was performed as in (24 (link)). The primary antibodies for phospho-ERK, total-ERK, phospho-Histone 3, Mcl-1, BIM and total MEK, were from Cell Signaling Technology. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Sigma Aldrich) was used as a loading control to demonstrate even protein loading. The secondary antibodies; goat anti-rabbit IgG HRP and sheep anti-mouse IgG HRP were from Amersham/ GE Healthcare. For mouse xenograft experiments, tumor samples were harvested and immediately stored in RNAlater solution (Ambion) at 4°C before protein extraction.

Phadke M.S., Sini P, & Smalley K.S. (2015). The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression. Molecular cancer therapeutics, 14(6), 1354-1364.

+ Open protocol

+ Expand

EGF-induced ERK activation in HEK293-EBNA cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293-EBNA cells were transfected with pmGFP-H-ras(wt) alone or co-transfected with pmRFP-Gal-1 or pmRFP-N-Gal-1 using JetPRIME transfection reagent (Polyplus transfection). After 24 h cells, which were previously serum starved for 5 h, were stimulated with 100 ng/ml EGF (Sigma-Aldrich) for 0 min, 2 min, 5 min, 10 min, 15 min and 30 min and lysed using SDS lysis buffer. Cell lysates were separated by SDS-PAGE and blotted using pERK (Cell Signalling, #9106), total ERK (Cell Signalling, #9102) and β-actin (Sigma Aldrich, A1978) antibodies. The Chemidoc MP system (Bio-Rad Laboratories) was used to detect the band intensities, which were then quantified by densitometry in ImageJ software. Band intensities of pERK were normalized to the ones of total ERK. Averages from three different biological repeats were calculated.

Blaževitš O., Mideksa Y.G., Šolman M., Ligabue A., Ariotti N., Nakhaeizadeh H., Fansa E.K., Papageorgiou A.C., Wittinghofer A., Ahmadian M.R, & Abankwa D. (2016). Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering. Scientific Reports, 6, 24165.

+ Open protocol

+ Expand

Edaravone Attenuates Oxidative Stress

Check if the same lab product or an alternative is used in the 5 most similar protocols

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) was purchased from Mitsubishi Pharma Corporation (Tokyo, Japan). DEM, D-galactosamine (Gal), LPS, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were products from Sigma-Aldrich (St Louis, MO). PD98059 and staurosporine were products of Calbiochem (Billerica, MA). All cell culture consumables and reagents were bought from either Corning Incorporated (Corning, NY) or Gibco (Carlsbad, CA). Antibodies against catalase (CAT), SOD1, phosphorylated p38 MAPK at Thr180/Tyr182, total p38 MAPK, phosphorylated ERK at Thr202/Tyr204, total ERK, and β-actin were bought from Cell Signaling (Beverly, MA).

Zeng W., Xiao J., Zheng G., Xing F., Tipoe G.L., Wang X., He C., Chen Z.Y, & Liu Y. (2015). Antioxidant treatment enhances human mesenchymal stem cell anti-stress ability and therapeutic efficacy in an acute liver failure model. Scientific Reports, 5, 11100.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!