Anti phospho erk

Manufactured by Merck Group

Sourced in United States

Anti-phospho-ERK is a laboratory reagent that detects the phosphorylated form of extracellular signal-regulated kinase (ERK). It is used to analyze the activation status of the ERK signaling pathway in cells.

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Anti-ERK (13 citations) Phospho-ERK (10 citations) Anti-phospho-ERK antibody (4 citations) Mouse anti-phospho-ERK (2 citations)

13 protocols using anti phospho erk

Phosphorylation of ERK and p38

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The primary antibodies used: anti-phospho-ERK (Cat# M-9692) was purchased from Sigma Aldrich. Anti-p38 (Cat# 9212), anti-phospho-p38 (Cat# 9211) and anti-ERK (Cat# 9102) were purchased from Cell Signaling.

Kalfon R., Friedman T., Eliachar S., Shofti R., Haas T., Koren L., Moskovitz J.D., Hai T, & Aronheim A. (2019). JDP2 and ATF3 deficiencies dampen maladaptive cardiac remodeling and preserve cardiac function. PLoS ONE, 14(2), e0213081.

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FGF-2 Stimulation of Human Chondrocytes

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P0–P3 HACs were cultured as monolayers and transfected 36–48 h before lysis. When appropriate, HACs were stimulated with a 25 nM human FGF-2 (Prepotech, UK). The age and gender of the HACs donors used for each experiment are indicated in the figure legends. HACs were lysed in a radio-immunoprecipitation assay buffer (RIPA—150 mM NaCl, 10 mM Tris, pH 7.2, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid) and 10–30 μg total protein extract subjected to SDS-polyacrilamide gel electrophoresis and transferred to PVDF membranes (Merck-Millipore, Germany). Mouse monoclonal anti-Ago2 (clone2E12-1C9, Abnova, 1:1000 dilution), rabbit monoclonal anti-DUSP6 (EPR129Y, Abcam, 1:1000 dilution), rabbit monoclonal anti-phospho-ERK (Cat No. 05-797R, Sigma-Aldrich, 1:1000 dilution), rabbit polyclonal anti-ERK (Cat No. sc-292838, SantaCruz Biotechnologies, 1:500 dilution), mouse monoclonal anti-Tubulin (clone B-5-1-2, Sigma-Aldrich, 1:5000 dilution), HRP-conjugated anti-mouse IgG secondary (Cat No. NA931, GE Healthcare, 1:5000 dilution), and HRP-conjugated anti-rabbit IgG secondary (Cat No. NA934, GE Healthcare, 1:5000 dilution), antibodies were used. Proteins were visualized by ECL fluorography (GE Healthcare, Germany).

Martinez-Sanchez A., Lazzarano S., Sharma E., Lockstone H, & Murphy C.L. (2020). High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes. Life, 10(5), 58.

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Western Blot Analysis of ERK Activation

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MSCs at passage 3 and 6 were plated in 6-well plates at a density of 1 × 10⁵cells/cm² and starved in serum-free α-MEM medium for at least 6 h. Protein
lysis buffer (Bio-Rad, Hercules, CA, USA) was added, and thawed lysates were vortexed and
centrifuged. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel
electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked
by incubation with 5% wt/vol nonfat dry milk. Membranes were then incubated with anti-ERK,
anti-phospho-ERK, and β-actin (Sigma) Abs at the appropriate dilutions overnight at 4°C.
After incubation, the membranes were washed in Tris-buffered saline containing Tween-20
(TBST). Secondary antibody conjugated to horseradish peroxidase was added to the membranes
in 5% nonfat dry milk in TBST. The negative control was used as described previously. The
western blotting assay was performed at least 3 times independently, representative
results are shown.

Zhang H., Li Z.L., Su X.Z., Ding L., Li J, & Zhu H. (2018). Subchondral bone derived mesenchymal stem cells display enhanced osteo-chondrogenic differentiation, self-renewal and proliferation potentials. Experimental Animals, 67(3), 349-359.

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Mutational Profiling and Inhibitor Sensitivity of Melanoma Cell Lines

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SkMel28 cells (American Type Culture Collection; ATCC; Rockville, MD), A375, DM13, DM331, DM6, HT144, SKMel24, VMM12, VMM15, VMM17, VMM18, and VMM5A (kind gift from Dr. Craig Slingluff, University of Virginia) were propagated in RPMI Medium 1640 (Invitrogen, Grand Island, NY) supplemented with 5% or 10% fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA). All cultures were maintained in a humidified chamber at 37°C with 5% CO₂. OncoMap analysis was conducted at the Broad Institute to identify the mutational status of over 30 known oncogenes and tumor suppressor genes. The cell lines were authenticated by comparing the tumor mutation profile determined by OncoMap with published reports.
Antibodies were obtained from the following sources: anti-phosphoERK and anti-total ERK (Sigma-Aldrich), and anti-tubulin (Calbiochem, Gibbstown, NJ).
Sources of combination inhibitors are shown in Supplementary Table S1. PLX4720 was a gift from Plexxikon, RAF265 a gift from Novartis and PD325901 a gift from Pfizer.

Roller D.G., Capaldo B., Bekiranov S., Mackey A.J., Conaway M.R., Petricoin E.F., Gioeli D, & Weber M.J. (2015). Combinatorial drug screening and molecular profiling reveal diverse mechanisms of intrinsic and adaptive resistance to BRAF inhibition in V600E BRAF mutant melanomas. Oncotarget, 7(3), 2734-2753.

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Signaling Pathway Characterization Protocol

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Anti–TAK‐1 (catalog no. D94D7), anti–MKK‐4 (catalog no. 9152S), anti–phospho–MKK‐4T261 (catalog no. 9151S), anti‐IκB (catalog no. 4814S), anti–phospho‐IκB, Ser32/36 (catalog no. 9246), anti–phospho–p38‐T180/Y182 (catalog no. 9211S), anti–phospho–TAK‐1‐T184/187 (catalog no. 45085), anti–phospho–IKKα/β‐Ser176/180 (catalog no. 16A6), and anti–phospho–activating transcription factor 2 (ATF‐2)–Thr71 (catalog no. 9221) were from Cell Signaling Technology. Anti–phospho–JNK‐pTpY183/185 (catalog no. 44682G) was obtained from Invitrogen. Anti–T‐ERK (catalog no. sc‐94), anti–NF‐κB–p65 (catalog no. Sc‐372), Anti–TAK‐1 (catalog no. sc‐7162), and anti–phospho–TAK‐1‐S192 (catalog no. sc‐130219) were from Santa Cruz Biotechnology. Anti‐ubiquitin–Lys‐63–specific antibody (catalog no. 05‐1308) was from Millipore. Myelin basic protein (MBP; catalog no. M1891) and anti–phospho‐ERK (catalog no. M9692) were from Sigma. A ubiquitin chain restriction enzyme analysis (UbiCREST) kit (catalog no. K‐400) was obtained from R&D Systems, and γ³²P‐ATP was obtained from PerkinElmer. TAK‐1 inhibitor 5z‐7‐oxozeanol (catalog no. 3604) was from Tocris.

Ismail H.M., Didangelos A., Vincent T.L, & Saklatvala J. (2017). Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K63‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage. Arthritis & Rheumatology (Hoboken, N.j.), 69(3), 565-575.

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Phosphorylation Signaling Antibodies

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Anti-phospho-AMPKα Thr172 (#2535), phospho-TAK1 Thr184 (#4537), phospho-GSK-3α/β Ser21/9 (#9331), and phospho-Akt Ser473 (#9271) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-p70S6K Thr412 (#07–018) and phospho-FOXO Thr32 (#07–694) were purchased from Millipore (Billerica, MA, USA). Anti-phospho-ERK (#M8159) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-JNK (#44-682G) was purchased from Biosource (Carlsbad, CA, USA). Anti-phospho-p38 MAPK Thr180/Tyr182 (#10009177) was purchased from Cayman Chemical. Anti-GAPDH (#ab36840) was purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibody (#ALI4404) was purchased from Biosource. Rabbit anti-mouse secondary antibody (#A9044) was purchased from Sigma-Aldrich. Morpholinos were purchased from Gene Tools, LLC (Philomath, OR, USA).

Glennon E.K., Torrevillas B.K., Morrissey S.F., Ejercito J.M, & Luckhart S. (2017). Abscisic acid induces a transient shift in signaling that enhances NF-κB-mediated parasite killing in the midgut of Anopheles stephensi without reducing lifespan or fecundity. Parasites & Vectors, 10, 333.

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Protein Expression Analysis by Western Blot

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Total cellular proteins were extracted, quantified, and subjected to gel electrophoresis according to standard procedures as we described previously [49] (link). The antibodies used in our study, including anti-caspase 3 (#9662), anti-cleaved caspase 3 (#9661), anti-caspase 7 (#9494), anti-poly(ADP-ribose) polymerase (PARP) (P248; Sigma), anti-Bax (#2774), anti-Bcl-2 (#2872), anti-phospho-ERK (#9101), anti-ERK (#9102), anti-phospho-p38 (#4511), anti-p38 (#9212), anti-phospho-JNK (#9255), anti-JNK (#9252), and anti-actin (A5441; Sigma). Antibodies were obtained from Cell Signaling, Danvers, MA unless specified otherwise. The antigen-antibody complexes were visualized with by chemiluminescence with the Amersham ECL detection system (GE Healthcare, Piscataway, NJ).

Chang Y.C., Hsu Y.C., Liu C.L., Huang S.Y., Hu M.C, & Cheng S.P. (2014). Local Anesthetics Induce Apoptosis in Human Thyroid Cancer Cells through the Mitogen-Activated Protein Kinase Pathway. PLoS ONE, 9(2), e89563.

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Western Blot Analysis of Cell Signaling

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Cells were lysed in SDS sample buffer and analysed by standard Western-blotting protocols. The antibodies used were as follows: MITF clone C5 from Neomarkers, Lab Vision, Runcorn, UK; CDK2 (D-12), CDK4 (H-22) and ERK2 (C-14) from Santa-Cruz Biotechnolgy, Santa Cruz, CA, USA. Antibodies against p65, cleaved caspase3 and pIκBα were from Cell Signaling, Boston, MA and against BCL2 and p27 from BD Biosciences, Oxford, UK. Anti-phospho-ERK was from Sigma, UK.

Smith M.P., Sanchez-Laorden B., O’Brien K., Brunton H., Ferguson J., Young H., Dhomen N., Flaherty K.T., Frederick D.T., Cooper Z.A., Wargo J.A., Marais R, & Wellbrock C. (2014). The immune-microenvironment confers resistance to MAP kinase pathway inhibitors through macrophage-derived TNFα. Cancer discovery, 4(10), 1214-1229.

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Western Blot Analysis of Autophagy Signaling

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Total cell extracts preparation and Western blot analysis were performed as previously described [13 (link)]. The antibodies used were anti-BRAF and anti-AMPK (Santa Cruz Biotechnology, Dallas, TX, USA); anti-p62 (BD Biosciences, Franklin Lakes, NJ, USA); anti-ATG5, anti-phospho-LKB1 (S428), anti-LKB1, anti-phospho-ACC (S79), anti-phospho-AMPK (T172), anti-phosho-S6 (S235/236) and anti-phospho-ULK (S555) (Cell Signalling Technology, Danvers, MA, USA); anti-LC3, anti-phospho-ERK and anti-β-tubulin (Sigma, St. Louis, MO, USA); and peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark). For LC3-II/LC3-I ratio calculation, data densitometry of blots from different experiments was used to quantify the protein bands.

Jiménez-Mora E., Gallego B., Díaz-Gago S., Lasa M., Baquero P, & Chiloeches A. (2021). V600EBRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells. International Journal of Molecular Sciences, 22(11), 6033.

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Quantifying Arp2/3 Complex Localization

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PtK1 cells were grown on glass coverslips for 48 hours, fixed with 3.7% formaldehyde with 10 nM Caliculyn A, in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7). Cells were permeabilized and blocked with 0.2% Triton and 3% BSA. Cells were stained with purified mouse monoclonocal anti-phospho-ERK (Sigma), rabbit anti-ArpC2 (Millipore), and phalloidin-647 (Molecular Probes) in 1% BSA, 0.2% Tween-20 in TBS. Images were acquired on a Nikon Ti motorized inverted microscope with Yokogawa CSU-X1 spinning disk confocal, using 405, 488, 561nm and 647 solid state laser excitation and an Andor Clara cooled CCD camera controlled by Nikon Elements image acquisition software.
Cells were segmented by thresholding in the phalloidin-647 channel. Because the peak Arp2/3 fluorescence was in front of the phalloidin signal, the segmented masks were then dilated out 0.5 microns from the edge. These dilated masks were then applied to the 488 and 568 channels, and the signal intensities were integrated over the cell area (mask). The ARP2C-568 fluorescence intensity was further averaged in a 1 micron-thick band parallel to the cell edge (0.5 microns to 0.5 microns) and the percentage of total signal present in this band was averaged for each group. The standard error of the mean and a 95% confidence interval was graphed.

Mendoza M.C., Vilela M., Juarez J.E., Blenis J, & Danuser G. (2015). ERK reinforces actin polymerization to power persistent edge protrusion during motility. Science signaling, 8(377), ra47.

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