|Replacement Information||The recommended replacement for this product is Cat. No. ABS44|
Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Av, H, M, R, Sh, Xn, Ech||ICC, IP, WB||Rb||Affinity Purified||Polyclonal Antibody|
|Presentation||Immunoaffinity purified immunoglobulin in 0.07M Tris-glycine, pH 7.4, 0.105 M NaCl, 0.035% sodium azide as a preservative.|
|Application||Anti-MAP Kinase 1/2 (Erk1/2) Antibody, CT detects level of MAP Kinase 1/2 (Erk1/2) & has been published & validated for use in IC, IP & WB.|
|Safety Information according to GHS|
|Material Size||100 µg|
|Reference overview||Application||Species||Pub Med ID|
|Characterization of Aldh2 (-/-) mice as an age-related model of cognitive impairment and Alzheimer's disease.|
D'Souza, Y; Elharram, A; Soon-Shiong, R; Andrew, RD; Bennett, BM
Molecular brain 8 27 2015
The study of late-onset/age-related Alzheimer's disease (AD)(sporadic AD, 95% of AD cases) has been hampered by a paucity of animal models. Oxidative stress is considered a causative factor in late onset/age-related AD, and aldehyde dehydrogenase 2 (ALDH2) is important for the catabolism of toxic aldehydes associated with oxidative stress. One such toxic aldehyde, the lipid peroxidation product 4-hydroxynonenal (HNE), accumulates in AD brain and is associated with AD pathology. Given this linkage, we hypothesized that in mice lacking ALDH2, there would be increases in HNE and the appearance of AD-like pathological changes.Changes in relevant AD markers in Aldh2 (-/-) mice and their wildtype littermates were assessed over a 1 year period. Marked increases in HNE adducts arise in hippocampi from Aldh2 (-/-) mice, as well as age-related increases in amyloid-beta, p-tau, and activated caspases. Also observed were age-related decreases in pGSK3β, PSD95, synaptophysin, CREB and pCREB. Age-related memory deficits in the novel object recognition and Y maze tasks begin at 3.5-4 months and are maximal at 6.5-7 months. There was decreased performance in the Morris Water Maze task in 6 month old Aldh2 (-/-) mice. These mice exhibited endothelial dysfunction, increased amyloid-beta in cerebral microvessels, decreases in carbachol-induced pCREB and pERK formation in hippocampal slices, and brain atrophy. These AD-associated pathological changes are rarely observed as a constellation in current AD animal models.We believe that this new model of age-related cognitive impairment will provide new insight into the pathogenesis and molecular/cellular mechanisms driving neurodegenerative diseases of aging such as AD, and will prove useful for assessing the efficacy of therapeutic agents for improving memory and for slowing, preventing, or reversing AD progression.
|High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts.|
Rudisch, A; Dewhurst, MR; Horga, LG; Kramer, N; Harrer, N; Dong, M; van der Kuip, H; Wernitznig, A; Bernthaler, A; Dolznig, H; Sommergruber, W
PloS one 10 e0124283 2015
We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.
|Proximity-dependent initiation of hybridization chain reaction.|
Koos, B; Cane, G; Grannas, K; Löf, L; Arngården, L; Heldin, J; Clausson, CM; Klaesson, A; Hirvonen, MK; de Oliveira, FM; Talibov, VO; Pham, NT; Auer, M; Danielson, UH; Haybaeck, J; Kamali-Moghaddam, M; Söderberg, O
Nature communications 6 7294 2015
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.
|MicroRNA-132 enhances transition from inflammation to proliferation during wound healing.|
Li, D; Wang, A; Liu, X; Meisgen, F; Grünler, J; Botusan, IR; Narayanan, S; Erikci, E; Li, X; Blomqvist, L; Du, L; Pivarcsi, A; Sonkoly, E; Chowdhury, K; Catrina, SB; Ståhle, M; Landén, NX
The Journal of clinical investigation 125 3008-26 2015
Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.
|Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.|
Johlfs, MG; Gorjala, P; Urasaki, Y; Le, TT; Fiscus, RR
PloS one 10 e0132105 2015
Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.
|NMDA receptor- and ERK-dependent histone methylation changes in the lateral amygdala bidirectionally regulate fear memory formation.|
Gupta-Agarwal, S; Jarome, TJ; Fernandez, J; Lubin, FD
Learning & memory (Cold Spring Harbor, N.Y.) 21 351-62 2014
It is well established that fear memory formation requires de novo gene transcription in the amygdala. We provide evidence that epigenetic mechanisms in the form of histone lysine methylation in the lateral amygdala (LA) are regulated by NMDA receptor (NMDAR) signaling and involved in gene transcription changes necessary for fear memory consolidation. Here we found increases in histone H3 lysine 9 dimethylation (H3K9me2) levels in the LA at 1 h following auditory fear conditioning, which continued to be temporally regulated up to 25 h following behavioral training. Additionally, we demonstrate that inhibiting the H3K9me2 histone lysine methyltransferase G9a (H/KMTs-G9a) in the LA impaired fear memory, while blocking the H3K9me2 histone lysine demethylase LSD1 (H/KDM-LSD1) enhanced fear memory, suggesting that H3K9me2 in the LA can bidirectionally regulate fear memory formation. Furthermore, we show that NMDAR activity differentially regulated the recruitment of H/KMT-G9a, H/KDM-LSD1, and subsequent H3K9me2 levels at a target gene promoter. This was largely regulated by GluN2B- but not GluN2A-containing NMDARs via ERK activation. Moreover, fear memory deficits associated with NMDAR or ERK blockade were successfully rescued through pharmacologically inhibiting LSD1, suggesting that enhancements of H3K9me2 levels within the LA can rescue fear memory impairments that result from hypofunctioning NMDARs or loss of ERK signaling. Together, the present study suggests that histone lysine methylation regulation in the LA via NMDAR-ERK-dependent signaling is involved in fear memory formation.
|Paradoxical results in perturbation-based signaling network reconstruction.|
Prabakaran, S; Gunawardena, J; Sontag, E
Biophysical journal 106 2720-8 2014
Mathematical models are extensively employed to understand physicochemical processes in biological systems. In the absence of detailed mechanistic knowledge, models are often based on network inference methods, which in turn rely upon perturbations to nodes by biochemical means. We have discovered a potential pitfall of the approach underpinning such methods when applied to signaling networks. We first show experimentally, and then explain mathematically, how even in the simplest signaling systems, perturbation methods may lead to paradoxical conclusions: for any given pair of two components X and Y, and depending upon the specific intervention on Y, either an activation or a repression of X could be inferred. This effect is of a different nature from incomplete network identification due to underdetermined data and is a phenomenon intrinsic to perturbations. Our experiments are performed in an in vitro minimal system, thus isolating the effect and showing that it cannot be explained by feedbacks due to unknown intermediates. Moreover, our in vitro system utilizes proteins from a pathway in mammalian (and other eukaryotic) cells that play a central role in proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis. This pathway is the perturbation target of contemporary therapies for various types of cancers. The results presented here show that the simplistic view of intracellular signaling networks being made up of activation and repression links is seriously misleading, and call for a fundamental rethinking of signaling network analysis and inference methods.
|Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells.|
Spampatti, M; Vlotides, G; Spöttl, G; Maurer, J; Göke, B; Auernhammer, CJ
World journal of gastroenterology 20 10038-49 2014
To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro.Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student's t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P less than 0.05.Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P less than 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P less than 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P less than 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1, serine/threonine kinase P70S6K and S6 ribosomal protein and inhibited glycogen synthase kinase 3 activity. We observed the (compensatory) activation of tuberous sclerosis 2, the serine/threonine specific protein kinase AKT and extracellular signal-regulated kinases.Aspirin demonstrates promising anticancer properties for NETs in vitro. Further preclinical and clinical studies are needed.
|The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling, and relationship to metabotropic glutamate receptors.|
Golubeva, AV; Zhdanov, AV; Mallel, G; Dinan, TG; Cryan, JF
Physiological reports 2 e00260 2014
Abstract Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Despite this, there is an unmet medical need in terms of effective diagnostics and treatment. Animal models are crucial in this endeavor. Systemic chronic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder pain. However, molecular mechanisms underlying the pathogenesis of this model are lacking. Here, we show that mice, subjected to repetitive systemic injections of CYP, developed mild inflammatory response in bladder tissue characterized by submucosal edema, moderate increase in proinflammatory cytokine gene expression, and mastocytosis. No signs of massive inflammatory infiltrate, tissue hemorrhages, mucosal ulcerations and urothelium loss were observed. Instead, CYP treatment induced urothelium hyperplasia, accompanied by activation of proliferative signaling cascades, and a decrease in the expression of urothelium-specific markers. Metabotropic glutamate (mGlu) receptors have been implicated in chronic pain disorders. CYP administration induced differential changes in mGlu receptors mRNA levels in bladder tissue, without affecting gene expression at spinal cord level, pointing to the potential link between peripheral mGlu receptors and inflammation-induced bladder malfunction and hyperalgesia. Taken together, these data indicate that chronic CYP treatment in mice is a model of PBS mostly relevant to the major, nonulcerative subtype of the syndrome, characterized by a relatively unaltered mucosa and a sparse inflammatory response. This model can help to elucidate the pathogenetic mechanisms of the disease.
|Distinct antifibrogenic effects of erlotinib, sunitinib and sorafenib on rat pancreatic stellate cells.|
Elsner, A; Lange, F; Fitzner, B; Heuschkel, M; Krause, BJ; Jaster, R
World journal of gastroenterology 20 7914-25 2014
To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their action.Cultured rat PSC were exposed to SMI. Cell proliferation and viability were assessed employing 5-bromo-2'-deoxyuridine incorporation assay and flow cytometry, respectively. 2-Deoxy-2-[(18)F] fluoroglucose ((18)F-FDG) uptake was measured to study metabolic activity. Exhibition of the myofibroblastic PSC phenotype was monitored by immunofluorescence analysis of α-smooth muscle actin (α-SMA) expression. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Transforming growth factor-β1 (TGF-β1) levels in culture supernatants were quantified by ELISA.All three SMI inhibited cell proliferation and (18)F-FDG uptake in a dose-dependent manner and without significant cytotoxic effects. Furthermore, additive effects of the drugs were observed. Immunoblot analysis showed that sorafenib and sunitib, but not erlotinib, efficiently blocked activation of the AKT pathway, while all three drugs displayed little effect on phosphorylation of ERK1/2. Cells treated with sorafenib or sunitinib expressed less interleukin-6 mRNA as well as less collagen type 1 mRNA and protein. Sorafenib was the only drug that also upregulated the expression of matrix metalloproteinase-2 and reduced the secretion of TGF-β1 protein. All three drugs showed insignificant or discordant effects on the mRNA and protein levels of α-SMA.The tested SMI, especially sorafenib, exert inhibitory effects on activated PSC, which should be further evaluated in preclinical studies.
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