P mek1 2s217 221

Manufactured by Cell Signaling Technology

Sourced in United States

P-MEK1/2S217/221 is an antibody that detects phosphorylated MEK1/2 at serine 217/221. MEK1/2 is a key protein kinase in the MAPK/ERK signaling pathway. This antibody can be used to detect the activation state of MEK1/2 in various cellular assays.

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P-MEK1 (5 citations)

9 protocols using p mek1 2s217 221

Western Blot Analysis of Signaling Pathways in Purified Immune Cells

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Purified B cells or T-cells were lysed using 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 μg/ml leupetin, 1% Triton X-100, 1 mM PMSF and 1 mM Na₃VO₄. Total protein was quantified by the Bradford assay, and 10 μg was loaded per lane onto SDS-PAGE gels. The following primary Abs were obtained from Cell Signaling Technology: p-STAT3^S727 (#9134), P-Erk1,2^T202/Y204 (#4376), Erk1,2 (#4695), p-NF-κB^S536 (#3031), p-MEK1/2^S217/221 (#9121), MEK1/2 (#9122), AKT (#9272), p-AKT^T308 (#9275), α-tubulin (#2144). β-actin (Advanced Immunochemical Inc, catalog # RGM2), Bcl-2 (Santa Cruz Biotechnology, Inc., # sc-23960). Antibodies to Nrf2 (ab71890), NQO1 (ab2346), GCLC (ab53179) and GCLM (ab81445) were purchased from Abcam. HRP-conjugated secondary antibodies and the ECL-plus detection kit (Amersham) were used to develop the blot. For western blot analysis of purified B and T-cells, cells were pooled from 3-4 mice for each lane. The respective band intensities were measured using ImageJ software version 1.37 (http://rsb.info.nih.gov/ij), and normalized against the corresponding β-actin or α-tubulin levels. Where samples from different strains were compared, normalized band intensities were expressed as ratios, relative to the corresponding B6 levels.

Wu T., Ye Y., Min S.Y., Zhu J., Khobahy E., Zhou J., Yan M., Hemachandran S., Pathak S., Zhou X.J., Andreeff M, & Mohan C. (2014). Targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid prevents murine lupus nephritis. Arthritis & rheumatology (Hoboken, N.J.), 66(11), 3129-3139.

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Antibody Generation and Immunoblotting Protocol

Cited 1 time

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Rabbit anti-TNS1 antibody was generated against human TNS1 aa1328–1339 peptide^{16 (link)}. Antibodies against E-cadherin (#9121), p Mek1/2(S221) (#2338), p Mek1/2(S217/221) (#9154), Mek1/2 (#9122), p ERK1/2(T202Y204) (#9101), ERK1/2 (#9194), pAkt(S473) (#9271), Akt (#9272), p GSK3β(S9) (#5558), and GSK3β (#12456) were from Cell Signaling Technology. Anti-GAPDH (#CB1001) was from Millipore. Anti-Vinculin (#693291) was from MP Biomedical. Anti-mouse or rabbit IgG HRP conjugated secondary antibodies (#7076, #7074) were from Cell Signaling Technology. Alexa Fluor secondary antibodies (488/594) and Alexa Fluor 488/594 Phalloidin were from Thermo Scientific and anti-GP135/Podocalyxin (#3F2/D8) was from the Developmental Studies Hybridoma Bank at the University of Iowa. VECTASHIELD antifade DAPI mounting solution was from Vector Labs. Mek inhibitors CI-1040 (#S1020), selumetinib (#S1008), trametinib (#S2673) were from Selleckchem, and PD98059 (#9900), U0126 (#9903) were from Cell Signaling Technology.

Wu Z.Y., Chiu C.L., Lo E., Lee Y.R., Yamada S, & Lo S.H. (2019). Hyperactivity of Mek in TNS1 knockouts leads to potential treatments for cystic kidney diseases. Cell Death & Disease, 10(12), 871.

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Protein Expression and Immunoblotting Analysis

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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were sonicated for 15 sec., centrifuged at 18k × g for 10 min. at 4°C, and protein in supernatants was quantified using BCA assay (Pierce). Reduced/denatured lysates were separated by SDS-PAGE, and proteins were transferred to nitrocellulose. Even protein loading across lanes was confirmed with Ponceau S staining. Blots were probed with antibodies against P-REX1 (Abcam), HA, HER3 (Santa Cruz), Rac1 (Cytoskeleton), p-AKT_S473, p-AKT_T308, AKT, p-S6_S240/4, p-ERK1/2_T202/Y204, p-MEK1/2_S217/221, p-IGF-1Rβ_Y1135/6/p-InsRβ_Y1150/1, IGF-1R, IRS-1, p-HER3_Y1289, or Actin (Cell Signaling). HRP-labeled secondary antibodies (GE Healthcare) and ECL substrate (Pierce) were used for signal detection.

Dillon L.M., Bean J.R., Yang W., Shee K., Symonds L.K., Balko J.M., McDonald W.H., Liu S., Gonzalez-Angulo A.M., Mills G.B., Arteaga C.L, & Miller T.W. (2014). P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT, and MEK/ERK signaling in breast cancer. Oncogene, 34(30), 3968-3976.

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Protein Expression and Immunoblotting Analysis

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Spheroid Culture and Signaling Pathway Analysis

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Spheroids in suspension or single cell-derived spheroids embedded in Matrigel GFR (5 × 10² cells in 7 μL of Matrigel GFR) were cultured for 7 days in the CTOS organoid medium. Western blotting analyses were performed⁵⁵ (link) using the following antibodies; The total AKT (#2920), pAKT-S473 (#9271), MEK1/2 (#9122), pMEK1/2-S217/221 (#9121), ERK1/2 (#9107), pERK1/2-T202/Y204 (#4370), GAPDH (#2118), S6 (#2212), p-S6 (#2211), c-Myc (#5605), and NICD (#4147) antibodies were obtained from Cell Signaling Technology. MSI1 (ab52865) antibody was obtained from Abcam. Secondary anti-rabbit (#7074) and -mouse (#7076) antibodies were obtained from Cell Signaling Technology. All antibodies were used at the producer’s suggested concentrations. For the DAPT treatment experiments, C45-4L spheroids were treated with 50 μM of DAPT (Abcam, ab120633) or 0.1% of DMSO (Millipore Sigma, D5879) as control, cultured overnight, and subjected to Western blotting analysis.

Coppo R., Kondo J., Iida K., Okada M., Onuma K., Tanaka Y., Kamada M., Ohue M., Kawada K., Obama K, & Inoue M. (2023). Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity. iScience, 26(2), 105962.

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Western Blot Analysis of Signaling Proteins

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RIPA buffer (#899900; Thermo Scientific) supplemented with protease/phosphatase inhibitors (#5872S; Cell Signaling) and EDTA solution (0.5mM [final]) was used to lyse cells. Membranes were probed overnight in 5% BSA at 4°C with gentle rocking with antibodies at manufacturers’ recommended dilutions. PLK3 (#4896), pMEK1/2^S217/221 (#9121S) and MEK1/2 (#4694S) antibodies were obtained from Cell Signaling. pERK^Y204 (#SC-7383), ERK1/2 (#SC-292838), beta-actin (SC-47778) and tubulin (#SC-8035) antibodies were obtained from Santa Cruz Biotechnology. GAPDH (#AM4300) antibody was obtained from Ambion. GFP antibody (#ab290) was obtained from Abcam.

Babagana M., Kichina J.V., Slabodkin H., Johnson S., Maslov A., Brown L., Attwood K., Nikiforov M.A, & Kandel E.S. (2019). The role of Polo-like kinase 3 in the response of BRAF-mutant cells to targeted anti-cancer therapies.. Molecular carcinogenesis, 59(1), 5-14.

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Protein Expression Analysis of Frozen Tumors

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Frozen tumor pieces were thawed, washed 2X with cold PBS and pestered to lyse in lysis buffer (50 mM Tris–HCl pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton-x-100, 50 mM NaF) supplemented with protease and phosphatase inhibitor cocktails (Bimake, Houston, TX, USA). Lysates were mixed with 4X Laemmli loading buffer and subjected to SDS-PAGE gel electrophoresis and Western blotting. The primary antibodies used were as following: pAkt S473 (#4060), p MEK1/2 S217/221 (#9121), NF-κB p65 (#6956), pPI3K p85 Y458/p55 Y199 (#4228), PI3K p85α (#13666), Rac-1 (#4651), p STAT3 Y705 (#9145), STAT3 (#9139), Wnt5a/b (#2530) (Cell Signaling Technology, Danvers, MA, USA); Akt (#sc-5298), Bcl-2 (#sc-7382), MEK1/2 (#sc-6250), β-tubulin (#sc-166,729) (Santa Cruz, Dallas, TX, USA); ROR1 6D4 (Dr. Riddel lab, ref. Balakrishana et al. 2016); ROR2 (#565550, BD Biosciences, San Jose, CA, USA). Secondary antibodies: IRDye® 800CW Donkey anti-Mouse IgG, or IRDye® 680RD Donkey anti-Rabbit IgG (LI-COR, Lincoln, NE, USA). Blots were scanned and quantified using Odyssey CLx and Image Studio software (LI-COR). Protein quantification was normalized to β-tubulin expression level for each sample.

Veskimäe K., Scaravilli M., Niininen W., Karvonen H., Jaatinen S., Nykter M., Visakorpi T., Mäenpää J., Ungureanu D, & Staff S. (2018). Expression Analysis of Platinum Sensitive and Resistant Epithelial Ovarian Cancer Patient Samples Reveals New Candidates for Targeted Therapies. Translational Oncology, 11(5), 1160-1170.

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Western Blot Analysis of Signaling Pathways

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After seeding and drug treatments, cells were washed with cold PBS and
lysed in RIPA buffer (Pierce #89901) plus phosphatase and protease inhibitors
(Thermo Scientific #1861277, #1861278). Lysates were cleared by centrifugation
at 14000rpm at 4°C and quantified using BCA method (Pierce #23224).
Samples were prepared using LDS+Reducing agent Novex buffers (Invitrogen
#NP0008, #NP0009). 10 to 20μg of lysates were loaded and run on NuPageTM
4–12% Bis-Tris gels (ThermoFisher #NP0321BOX) followed by transfer to
nitrocellulose membranes (Biorad #1620233). Membranes were incubated over night
with the indicated antibodies, washed and incubated again for 45 minutes with
anti-rabbit or anti-mouse secondary antibodies. Detection was performed using
Immobilion Western (Millipore #WBKLS0500).
Primary antibodies were obtained from Cell Signaling Technology and were
used at a concentration of 1:1000: p-Akt S473 (#9271), p-MEK1/2 S217/221
(#9154), p-p44/42 MAPK T202/204 (#9101), Total MEK1/2 (#9122), Total ERK1/2
(#9102), Total AKT (#9272), Total EGFR (#2232S), phospho-EGFR Tyr1068 (#3777S)
and Vinculin (#13901S). DUSP6 antibody was purchased from Abcam (#ab54940) and
used at 1:500 dilution. CyclinD1 antibody was purchased from Santa Cruz
(#sc-718) and used at 1:1000 dilution. Densitometry analyses were performed
using ImageJ software.

Amodio V., Yaeger R., Arcella P., Cancelliere C., Lamba S., Lorenzato A., Arena S., Montone M., Mussolin B., Bian Y., Whaley A., Pinnelli M., Murciano-Goroff Y.R., Vakiani E., Valeri N., Liao W.L., Bhalkikar A., Thyparambil S., Zhao H.Y., De Stanchina E., Marsoni S., Siena S., Bertotti A., Trusolino L., Li B.T., Rosen N., Di Nicolantonio F., Bardelli A, & Misale S. (2020). EGFR blockade reverts resistance to KRAS G12C inhibition in colorectal cancer. Cancer discovery, 10(8), 1129-1139.

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Molecular Mechanism of Vemurafenib Response

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Vemurafenib was purchased from ChemieTek. Sunitinib, imatinib, crenolanib and LDE225 were purchased from Selleck Chemicals LLC. MTT was purchased from Sigma. p-AKT (Ser473)-, AKT-, p-PI3K p85 (γ458)-, p-CRAF(S289/296/301)-, p-MEK 1/2 (S217/221)-, p-ERK 1/2 (Thr202/Tyr204)-, ERK1/2-, PDFGRβ-, p-PDGFRα-, PDGFRα-, PTEN-, VEGFR2-, Cleaved Caspase-3 (Asp175)-, p-Histone H3 (Ser10)-, Gli1- and β-actin-specific monoclonal antibodies (mAbs) were purchased from Cell Signaling Technology. The calnexin-specific mAb TO-5 was developed and characterized as described [55 (link)]. PDGFRα-specific shRNA and GFP-shRNA were provided by the- Vector Core Facility of the University of Pittsburgh Cancer Institute.

Sabbatino F., Wang Y., Wang X., Flaherty K.T., Yu L., Pepin D., Scognamiglio G., Pepe S., Kirkwood J.M., Cooper Z.A., Frederick D.T., Wargo J.A., Ferrone S, & Ferrone C.R. (2014). PDGFRα up-regulation mediated by sonic hedgehog pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation. Oncotarget, 5(7), 1926-1941.

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