Mouse anti mek1 2

Manufactured by Cell Signaling Technology

Sourced in United States

Mouse anti-MEK1/2 is a primary antibody that specifically recognizes Mitogen-Activated Protein Kinase Kinase 1 and 2 (MEK1/2). MEK1/2 are key enzymes in the MAPK/ERK signaling pathway, which is involved in the regulation of various cellular processes.

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Close spelling variants of this product

MEK1/2 (167 citations) Anti-MEK1/2 (56 citations) Anti-MEK1 (8 citations)

5 protocols using mouse anti mek1 2

Transient Transfection of Cell Lines

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HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections were performed with Transfectin reagent (Bio-Rad, 1703352). The lung cancer cell lines A549 (ATCC CCL-185) and H1666 (ATCC CRL-5885) were grown in Roswell Park Memorial Institute media (RPMI), the melanoma cell line A2058 (ATCC CRC-11147) in DMEM. The media was supplemented with 10% FBS and 10 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (Hepes). Quail embryo fibroblasts (QEF) were grown in Avian cell culture medium. DNA transfection was mediated using the calcium phosphate method. Primary antibodies used were the mouse anti-Rluc antibody directed against Rluc-F[1] (Chemicon, #MAB4410), mouse anti–HA-tag (Covance, MMS-10P), mouse anti-BRAF (Santa Cruz, F-7: sc-5284), mouse anti-MEK1/2 (Cell Signaling, 4684S), rabbit phospho-MEK1/2 (Ser217/221) (Cell Signaling, 9154), and rabbit anti–P-ERK1/2 (Cell Signaling, 9101).

Mayrhofer J.E., Enzler F., Feichtner A., Röck R., Fleischmann J., Raffeiner A., Tschaikner P., Ogris E., Huber R.G., Hartl M., Schneider R., Troppmair J., Torres-Quesada O, & Stefan E. (2020). Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies. Proceedings of the National Academy of Sciences of the United States of America, 117(49), 31105-31113.

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Western Blot Analysis of RAS/MAPK Signaling

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Example 16

For immunoblot analysis experiments, cells rinsed trice with ice-cold phosphate-buffered saline (PBS) were lysed on ice, with ice-cold TNE buffer, supplemented with Halt protease and phosphatase inhibitors (Thermo Scientific), and centrifuged at 15,000 g for 15 minutes to collect whole-cell lysates. Protein concentration was measured with the BCA protein assay (Pierce). Thirty micrograms of total protein per sample were loaded into 4%-12% NuPAGE Bis-Tris gradient gels (Life Technologies) and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The following antibodies were used for immunoblotting: mouse monoclonal anti-KRAS (Sigma WH0003845M1, clone 3B10-2F2), mouse anti-RAS (Thermo 1862335), rabbit anti-pERK1/2 (T202/Y204; Cell Signaling Technology 4370), mouse anti-ERK1/2 (Cell Signaling Technology 4696), rabbit anti-p-MEK1/2 (S217/221; Cell Signaling Technology 9154), mouse anti-MEK1/2 (Cell Signaling Technology 4694), rabbit anti-p-AKT (S473; Cell Signaling Technology 4060), mouse anti-AKT (Cell Signaling Technology 2920). Vinculin (rabbit anti-vinculin, Cell Signaling Technology 4650) was used as a loading control. Primary antibodies were detected with fluorescence-conjugated (LI-COR) secondary antibodies.

US11358940B2. K-Ras modulators (2022-06-14). THE REGENTS OF THE UNIVERSITY OF CALIFORNIA [US], LEIDOS BIOMEDICAL RESEARCH, INC. [US]. Inventors: Frank McCormick [US], Adam Renslo [US], David Turner [US], Anna E. Maciag [US], Marcin Dyba [US], Elizabeth D. Vo [US], Joseph Saavedra [US], Vandana Kumari [US].

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Western Blot Analysis of Signaling Pathways

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Primary antibodies: mouse anti-PARP-1, rabbit anti-phospho-Ser473 Akt, rabbit anti-phospho-Thr308 Akt, rabbit anti-Akt, rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-MEK1/2, mouse anti-MEK1/2, mouse anti-p21waf1, rabbit anti-p27, were from Cell Signaling Technologies (Boston, MA, USA). Mouse anti-human GAPDH and rabbit anti-cyclin A were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies: horseradish peroxidase (HRP)—conjugated goat anti-rabbit and goat anti-mouse IgG (H + L) antibodies were from Jackson (Baltimore Pike West Grove, PA, USA). EZ-ECL enhanced chemiluminescence detection kit, RPMI1640 medium, L-glutamine, fetal bovine serum, trypsin, antibiotics and phosphate buffered saline (PBS) were from Biological Industries (Beit-Ha-Emek, Israel). Propidium iodide, phosphatase inhibitor cocktails 2 and 3, sulphorodamine B (SRB), trichloroacetic acid and acetic acid were from Sigma Aldrich (St. Louis, MO, USA). ZSTK474, Selumetinib and AEW-541 were from Selleckchem (Houston, TX, USA). Complete mini protease inhibitor cocktail, RNAse A and Triton X-100 were from Roche Diagnostics Gmbl (Mannheim, Germany).

Cohen Z., Maimon Y., Samuels N., Brand H., Sulkes A., Brenner B, & Berger R. (2022). Strong Synergic Growth Inhibition and Death Induction of Cancer Cells by Astragalus membranaceus and Vaccaria hispanica Extract. Cancers, 14(23), 5833.

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Transfection Protocols for Cancer Cell Lines

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HEK293, A375, and SW480 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections were performed with TransFectin reagent (Bio-Rad, #1703352) or jetPRIME (Polyplus). Primary antibodies used were mouse anti-Rluc antibodies (Chemi-Con, #MAB4400 versus Rluc-F[2] and #MAB4410 versus Rluc-F[1]), mouse anti–HA-tag (Covance, #MMS-10P), mouse anti-FLAG (Sigma-Aldrich, #F3165), mouse anti-V5 (Invitrogen, #R9302), mouse anti-BRAF [Santa Cruz Biotechnology, sc-5284 (#F-7) and sc-166 (#C-19)], rabbit anti-CRAF (Cell Signaling Technology, #9422), mouse anti-RAS (Thermo Fisher Scientific, #MA1-012X), mouse anti-GFP (green fluorescent protein) (Roche, #11814460001), mouse anti-MEK1/2 (Cell Signaling Technology, #4684S), rabbit anti–P-MEK1/2 (Ser²¹⁷/Ser²²¹) (Cell Signaling Technology, #9154), rabbit anti–P-ERK1/2 (Cell Signaling Technology, #9101), rabbit anti-ERK1/2 (Cell Signaling Technology, #4696S), and mouse anti-PKA RIIβ (BD Biosciences, #61062).

Röck R., Mayrhofer J.E., Torres-Quesada O., Enzler F., Raffeiner A., Raffeiner P., Feichtner A., Huber R.G., Koide S., Taylor S.S., Troppmair J, & Stefan E. (2019). BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS. Science Advances, 5(8), eaav8463.

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Antibody and Knockdown Assay Protocol

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The following antibodies were used: rabbit anti-eIF4E2 (4EHP) (Genetex, GTX103977), mouse anti-eIF4E (BD Biosciences, 610270), rabbit anti-eIF4ENIF1 (4E-T; abcam, ab55881), rabbit anti-DDX6 (Bethyl Laboratories, A300-460A), rabbit anti-CNOT1 (Proteintech, 14276–1-AP), mouse anti-α-Tubulin (Santa Cruz, sc-23948), mouse anti-β-actin (Sigma, A5441), mouse anti-Flag (Sigma, F3165), rabbit anti-HA (Sigma, H6908), mouse anti-V5 tag (Invitrogen, R960-25), rabbit anti-PARP (Cell Signaling Cat# 9532S), rabbit anti-DUSP6 (abcam Cat# ab76310), rabbit anti-DUSP7 (abcam Cat# ab100921),), rabbit anti-CNOT9 (RQCD1) (Proteintech Cat# 22503–1-AP), mouse GAPDH (Santa Cruz, sc-32233), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Cat#4370), mouse anti-MEK1/2 (Cell Signaling Cat# 4694S), rabbit anti-phospho-MEK1/2 (Ser217/221; Cell Signaling Cat# 9121S), rabbit anti-phospho-RPS6 (Ser240/244) (Cell Signaling Cat# 2215), and mouse anti-RPS6 (C-8).
The following siRNA and shRNAs were used: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05), 4EHP siRNA SMARTpool (Dharmacon, L-019870–01), eIF4ENIF1 (4E-T) siRNA SMARTpool (Dharmacon, L-013237–01), CNOT1 siRNA SMARTpool (Dharmacon, L-015369–01-0005), CNOT9 siRNA SMARTpool (Dharmacon, L-019972–00), Non-Targeting shRNA Controls (Sigma, SHC002), and EIF4E2 shRNA (Sigma, TRCN0000152006).

Jafarnejad S.M., Chapat C., Matta-Camacho E., Gelbart I.A., Hesketh G.G., Arguello M., Garzia A., Kim S.H., Attig J., Shapiro M., Morita M., Khoutorsky A., Alain T., Gkogkas C.G., Stern-Ginossar N., Tuschl T., Gingras A.C., Duchaine T.F, & Sonenberg N. (2018). Translational control of ERK signaling through miRNA/4EHP-directed silencing. eLife, 7, e35034.

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