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Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay. (3905)

Notes: This article showed that expression of several immune response genes could be induced in lung adenocarcinoma H460 cells by treatment with farnesol and that this induction proceeds through an NF-κB pathway. To determine which MAPKs were involved in the activation of these genes, the authors used the MEK inhibitor U0126 and showed that it inhibited the expression of several of the immune response genes and specifically inhibited the phosphorylation of p65 on Ser²⁷⁶. (3904)

Notes: The authors of this study investigated the role of ERK1/2 kinase signaling in the preimplantation developmental events that precede compaction and blastocyst formation (2-cell to 8-cell stages). The MEK inhibitor U0126 was added to 2-cell stage mouse embryos, and the embryos were observed to arrest at the 4-cell stage. The arrest was reversible, but experiments indicated that ERK1/2 signaling is required for M-phase progression in early cell cycles during mouse development. (3906)

Notes: Mitogen-activated protein kinase phosphatase-1 (MKP-1) negatively regulates MAPK signaling and may have a role in cell survival in response to stress stimuli, including chemotherapeutics. This study investigates its role in cisplatin resistance. MAPK inhibitors U0126 (MEK inhibitor) and SB 203580 (p38 inhibitor) were used to determine whether the ERK pathway is required for cisplatin-induced MKP-1 expression in human lung cancer (H460) and human ovarian cancer (OVCAR3) cells. (3529)

Notes: The authors of this study sought to determine whether both MAP kinase and Cdk1/cyclin B kinase are required for mitotic entry and calcium signals in early sea urchin embryos. Treating embryos with MEK Inhibitor U0126 12 minutes after fertilization inhibited MAP kinase activity in the early embryos. Anti-ACTIVE^® MAPK pAb was used for immunoprecipitation of active MAP kinase at various times after fertilization. (3506)

Notes: Promega’s MEK Inhibitor U0126 was used to demonstrate specific CRE-mediated gene expression through Erk activation in mouse brains. An osmotic minipump pumped 250μM U0126 into animals before brain sections were dissected and examined for CRE-linked beta-galactosidase reporter in X-Gal histochemical studies.  (2795)

Notes: Vertebrate eggs arrest at meiosis II;  CSF (cytostatic factor) acts antatgonistically to anaphase promoting complex (APC) and maintains this arrest and prevents parthenogenetic activation in eggs prior to fertilization. Emil was identified as an inhibitor of APC. The authors used Mek Inhibitor U0126 to determine if the MAPK signaling pathway is required for maintaining the CSF arrest. Adding U0126 to CSF extracts led to inactivation of MAPK and p90^rsk but did not release extracts from the CSF arrest. The authors conclude that the MAPK pathway is not required to maintain CSF arrest. (2432)

Notes: Treatment of the human medulloblastoma MED283 cells with NGF causes a reduction is cell viability over a 72-hour period as judged by the CellTiter 96^® AQ_ueous Cell Proliferation Assay. The death was due to apoptosis. Neither the PD98059 inhibitor nor the MEK Inhibitor U0126 could prevent the apoptosis. The inhibitors (50µM and 10µM, respectively) were added to the media, which was changed every 12 hours during the 72-hour period. Cells with inhibitor and no NGF were not apoptotic, nor was there any noticeable decrease in cell viability. The data for the MEK Inhibitor U0126 Inhibitor is not shown. (1296)

Notes: The MEK Inhibitor U0126 was used to further define the role of MAPK activation in the timing of mitosis in a fertilized egg. The U0126 Inhibitor prevented MAPK activation and the calcium-induced H1 kinase activation and nuclear envelope breakdown occured approximately 30 minutes earlier than in control cells. It is also known that there is a transient activation of MAPK during mitosis following cdc2 activation. Addition of the U0126 Inhibitor did not change the cdc2 activation or mitosis and paralleled vehicle only controls. (0195)

Notes: The MEK Inhibitor U0126 was shown to inhibit MAPK phosphorylation completely and to a greater extent than PD98059. The inactivation had very little effect on neurite outgrowth. MAPK phosphorylation was determined with the Anti-ACTIVE^® MAPK pAb by Western blotting. (0297)

Notes: Cultured rat neurons were treated with BDNF with or without preincubation with 50uM U0126 MEK Inhibitor. The U0126 Inhibitor blocked both MAPK phosphorylation and CREB phosphorylation. The assay used to judge the phosphorylation was a immunohistochemical technique that used a confocal microscope and image processing software to measure the mean fluorescence from the microscope's field. (0536)

Notes: Primary mouse astrocytes were treated with an anti-Fas antibody, which induced expression of chemokines. Pretreatment of the cells with the U0126 MEK Inhibitor 30min prior to a 1hr treatment with the antibody reduced chemokine mRNA to basal levels. Neither oloumoucine or SB202190 (p38 inhibitor) duplicated the inhibition. (0818)

Notes: The MEK Inhibitor U0126 could interfere with the eIF4F assembly presumably through interference with phosphorylation of the components. The inhibitor blocked ERK phosphorylation in phorbol ester treatment with a 45-minute pretreatment with 50µM MEK Inhibitor U0126. Twenty micromolar MEK Inhibitor U0126 did not fully block phosphorylation. (1037)

Notes: The MEK Inhibitor U0126 was used to inhibit constitutive activity of Mek1 in order to determine which growth factors are necessary for C2 muscle cell line viability. U0126 was used at a 10 micromolar concentration. (2165)

Notes: The MEK Inhibitor U0126 was shown to inhibit MAPK activation in human eosinophils. IL-5-induced leukotriene C4 release was inhibited. The inhibitor was preincubated with the cells for 1 hour prior to addition of IL-5. Activation of MAPK was followed with the Anti-ACTIVE^® MAPK pAb via western blotting. (1467)

Notes: The protein aquaporin-5 is upregulated by osmotic shock in MLE-15 mouse lung epithelial cells. The shock causes an increase in ERK activation. The activation was blocked by the MEK Inhibitor U0126. The cells were treated with both inhibitor and activator for 15 minutes prior to lysis. (1049)

Notes: A primary cell line from rat subventricular zone which remains undifferentiated over time was developed as a model for MAPK activation. Cell were stimulated with 20 ng/ml bFGF and 20 ng/ml EGF (Promega) to activate MAPK. The MAPK pathway was inhibited with either U0126 (Promega) or  PD98059. Immunocytochemistry was performed with the Anti-ERK 1/2 pAb (1:100 dilution) to detect both active and inactive forms of MAPK proteins and with the Anti-ACTIVE™ MAPK pAb (1:100 dilution) to specifically detect the dually phosphorylated, active forms of MAPK. Cells were also immunostained with the neuron specific marker Anti-III-tubulin mAb (0.5 µg/ml). Cell proliferation was monitored with the CellTiter 96® AQueous One Solution Cell Proliferation Assay System. Apoptosis within the cell population was monitored using the DeadEnd™ Fluorometric TUNEL System. (2391)

Notes: HEK 293 cells stably expressing the mu-opioid receptor respond to agonists by MAPK phosphorylation. Activation of the MAPK was totally inhibited by the MEK Inhibitor U0126 at 100nM as judged by immunocytochemistry. The PD98059 required 20µM for the same inhibition. (0411)

Notes: The MEK Inhibitor U0126 was tested for its ability to block the increase in expression of the GABA-Aalpha6 subunit of the GABA-A receptor. The increased expression was judged by Northern blotting. Granule cells were cultured for 48hrs in serum-free media in the presence of BDNF and various concentrations of the U0126 Inhibitor. Total inhibition of the subunit's expression was noted with only 5-10µM of the inhibitor. The inhibitor PD98059 achieved the same level of inhibition with 25-50µM. Similar studies were performed with cells incubated for only 24hr with the BDNF and U0126 inhibitor. (1372)

Notes: Addition of exogenous H₂O₂ to cultured A549 cells causes a rapid increase in activated Erk as judged by the Anti-ACTIVE^® MAPK pAb via Western blotting. There is an inhibition of transcriptional activation of the amiloride-sensitive epithelial sodium channel α-subunit. The inhibition is reversed by 50µM U0126 MEK Inhibitor treatment 20 minutes prior to addition of H₂O₂ . Reporters were used for measurement of transcriptional activity and were produced in the pGL2-Promoter Vector. The reporter vectors were cotransfected into the A549 cells with the pRL-TK Vector at a 12:1 ratio. The luciferase activities were measured with the Dual-Luciferase^® Reporter Assay System. Transfections were accomplished with the ProFection^® Mammalian Transfection System-DEAE-Dextran. (0200)

Notes: The U0126 MEK Inhibitor inhibited both thapsigargin- and TPA-induced histamine production in a concentration dependent manner.Cells were cultured in 0.1 to 10µM U0126 Inhibitor for 24hr in the presence or absence of thapsigargin or TPA. Maximal inhibition was seen with 10µM inhibitor and ~50% inhibition at 1µM. (0397)

Notes: The MEK Inhibitor U0126 blocked both PMA-stimulated MAPK phosphorylation and paxillin phosphorylation in primary mouse splenocytes and thymocytes. MAPK phosphorylation was judged by immunoblotting extracts with the Anti-ACTIVE^® MAPK pAb, Rabbit, (pTEpY). The MEK Inhibitor U0126 was more effective than PD98059. (0853)

Notes: Serum-starved RAW264.7 macrophages were stimulated for 15 minutes with a laminin peptide, then extracts were made and analyzed for ERK phosphorylation. The treatment increased ERK phosphorylation; in other studies, it caused increases in urokinase and matrix metalloproteinase secretion into the media. The MEK Inhibitor U0126 could block ERK phosphorylation with a 30-minute preincubation of 10µM prior to the peptide treatment. The same pretreatment could block secretion of the two enzymes. (0935)

Notes: The pretreatment of PC12 cells with 20µM U0126 MEK Inhibitor for 20min prior to phorbol ester and IGF-1 treatment blocked the phosphorylation of Akt kinase. To determine the effect of PKC inhibitors on PKC activity in the PC12 cells after phorbol ester stimulation, lysates were prepared from various cellular fractions and analyzed for PKC activity with the SignaTECT^® PKC Assay System. Control values were obtained in the absence of the lipid activator solution and in the presence of the Myristoylated PKC Inhibitor Peptide. The greatest increase in PKC activity was found in the membrane fraction. (0058)

Notes: Both PMA and dibutryl cAMP (dBcAMP) are known to cause differentiation of cultured astrocytes into process-bearing stellate cells. The U0126 MEK Inhibitor blocks the formation of stellate cells by PMA but not by dBcAMP. The cells treated with dBcAMP had the same levels of Erk 1/2 as the PMA treated cells throughout the experiment but only PMA treated cells demonstrated activation of Erk 1/2 by dual phosphorylation. Thus, it is reasonable that the U0126 MEK Inhibitor could inhibit PMA-induced stellate formation. The level of total Erk 1 and 2 was determined by Western blotting with the Anti-Erk 1/2 pAb and the level of activated Erk 1/2 was determined with the Anti-ACTIVE^® MAPK pAb. Cells were treated for 20min with 10µM U0126 Inhibitor and treated with the stimulator for up to 6hr. The U0126 Inhibitor treatment blocked the stellate formation by PMA throughout the experiment. Titration of the U0126 Inhibitor was performed with 0.01 to 10µM and was compared to the PD98059 inhibitor. U0126 Inhibitor was completely inhibitory at 1 and 10µM and at 0.1µM was as inhibitory as 30µM PD98059. Protein kinase C inhibitors were also able to mimic the effect of the U0126 Inhibitor as demonstrated by the blockage of Erk 1/2 phosphorylation and stellate formation. The U0126 Inhibitor was also able to reverse the morphology of the stellate cells back to that of polygonal, undifferentiated cells. The reversal of morphology was also supported by the loss of activated Erk 1 and 2 as judged by Western blotting with the Anti-ACTIVE^® MAPK pAb. (2050)