Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R, Eq, Ca||WB||Ch||Affinity Purified||Polyclonal Antibody|
|Presentation||Purified chicken polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4, 150 mM NaCl) with 0.05% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8°C from date of receipt.|
|Material Size||100 µg|
Anti-GAPDH Antibody SDS
|Anti-GAPDH - 2424824||2424824|
|Anti-GAPDH - 1944115||1944115|
|Anti-GAPDH - 1986173||1986173|
|Anti-GAPDH - 2029184||2029184|
|Anti-GAPDH - 2105272||2105272|
|Anti-GAPDH - 2181288||2181288|
|Anti-GAPDH - 2276425||2276425|
|Anti-GAPDH - 2327505||2327505|
|Anti-GAPDH - 3022890||3022890|
|Anti-GAPDH - 3170836||3170836|
|Reference overview||Application||Species||Pub Med ID|
|Nkx2.2 and Nkx2.9 are the key regulators to determine cell fate of branchial and visceral motor neurons in caudal hindbrain.|
Jarrar, W; Dias, JM; Ericson, J; Arnold, HH; Holz, A
PloS one 10 e0124408 2015
Cranial motor nerves in vertebrates are comprised of the three principal subtypes of branchial, visceral, and somatic motor neurons, which develop in typical patterns along the anteroposterior and dorsoventral axes of hindbrain. Here we demonstrate that the formation of branchial and visceral motor neurons critically depends on the transcription factors Nkx2.2 and Nkx2.9, which together determine the cell fate of neuronal progenitor cells. Disruption of both genes in mouse embryos results in complete loss of the vagal and spinal accessory motor nerves, and partial loss of the facial and glossopharyngeal motor nerves, while the purely somatic hypoglossal and abducens motor nerves are not diminished. Cell lineage analysis in a genetically marked mouse line reveals that alterations of cranial nerves in Nkx2.2; Nkx2.9 double-deficient mouse embryos result from changes of cell fate in neuronal progenitor cells. As a consequence progenitors of branchiovisceral motor neurons in the ventral p3 domain of hindbrain are transformed to somatic motor neurons, which use ventral exit points to send axon trajectories to their targets. Cell fate transformation is limited to the caudal hindbrain, as the trigeminal nerve is not affected in double-mutant embryos suggesting that Nkx2.2 and Nkx2.9 proteins play no role in the development of branchiovisceral motor neurons in hindbrain rostral to rhombomere 4.
|hnRNP K coordinates transcriptional silencing by SETDB1 in embryonic stem cells.|
Thompson, PJ; Dulberg, V; Moon, KM; Foster, LJ; Chen, C; Karimi, MM; Lorincz, MC
PLoS genetics 11 e1004933 2015
Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs.
|Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.|
Kruiswijk, F; Hasenfuss, SC; Sivapatham, R; Baar, MP; Putavet, D; Naipal, KA; van den Broek, NJ; Kruit, W; van der Spek, PJ; van Gent, DC; Brenkman, AB; Campisi, J; Burgering, BM; Hoeijmakers, JH; de Keizer, PL
Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.Oncogene advance online publication, 17 August 2015; doi:10.1038/onc.2015.282.
|Follistatin promotes adipocyte differentiation, browning, and energy metabolism.|
Braga, M; Reddy, ST; Vergnes, L; Pervin, S; Grijalva, V; Stout, D; David, J; Li, X; Tomasian, V; Reid, CB; Norris, KC; Devaskar, SU; Reue, K; Singh, R
Journal of lipid research 55 375-84 2014
Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.
|Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity.|
Kreple, CJ; Lu, Y; Taugher, RJ; Schwager-Gutman, AL; Du, J; Stump, M; Wang, Y; Ghobbeh, A; Fan, R; Cosme, CV; Sowers, LP; Welsh, MJ; Radley, JJ; LaLumiere, RT; Wemmie, JA
Nature neuroscience 17 1083-91 2014
Acid-sensing ion channel 1A (ASIC1A) is abundant in the nucleus accumbens (NAc), a region known for its role in addiction. Because ASIC1A has been suggested to promote associative learning, we hypothesized that disrupting ASIC1A in the NAc would reduce drug-associated learning and memory. However, contrary to this hypothesis, we found that disrupting ASIC1A in the mouse NAc increased cocaine-conditioned place preference, suggesting an unexpected role for ASIC1A in addiction-related behavior. Moreover, overexpressing ASIC1A in rat NAc reduced cocaine self-administration. Investigating the underlying mechanisms, we identified a previously unknown postsynaptic current during neurotransmission that was mediated by ASIC1A and ASIC2 and thus well positioned to regulate synapse structure and function. Consistent with this possibility, disrupting ASIC1A altered dendritic spine density and glutamate receptor function, and increased cocaine-evoked plasticity, which resemble changes previously associated with cocaine-induced behavior. Together, these data suggest that ASIC1A inhibits the plasticity underlying addiction-related behavior and raise the possibility of developing therapies for drug addiction by targeting ASIC-dependent neurotransmission.
|CNS expression of murine fragile X protein (FMRP) as a function of CGG-repeat size.|
Ludwig, AL; Espinal, GM; Pretto, DI; Jamal, AL; Arque, G; Tassone, F; Berman, RF; Hagerman, PJ
Human molecular genetics 23 3228-38 2014
Large expansions of a CGG-repeat element (greater than 200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.
|Genetic suppression of transgenic APP rescues Hypersynchronous network activity in a mouse model of Alzeimer's disease.|
Born, HA; Kim, JY; Savjani, RR; Das, P; Dabaghian, YA; Guo, Q; Yoo, JW; Schuler, DR; Cirrito, JR; Zheng, H; Golde, TE; Noebels, JL; Jankowsky, JL
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 3826-40 2014
Alzheimer's disease (AD) is associated with an elevated risk for seizures that may be fundamentally connected to cognitive dysfunction. Supporting this link, many mouse models for AD exhibit abnormal electroencephalogram (EEG) activity in addition to the expected neuropathology and cognitive deficits. Here, we used a controllable transgenic system to investigate how network changes develop and are maintained in a model characterized by amyloid β (Aβ) overproduction and progressive amyloid pathology. EEG recordings in tet-off mice overexpressing amyloid precursor protein (APP) from birth display frequent sharp wave discharges (SWDs). Unexpectedly, we found that withholding APP overexpression until adulthood substantially delayed the appearance of epileptiform activity. Together, these findings suggest that juvenile APP overexpression altered cortical development to favor synchronized firing. Regardless of the age at which EEG abnormalities appeared, the phenotype was dependent on continued APP overexpression and abated over several weeks once transgene expression was suppressed. Abnormal EEG discharges were independent of plaque load and could be extinguished without altering deposited amyloid. Selective reduction of Aβ with a γ-secretase inhibitor has no effect on the frequency of SWDs, indicating that another APP fragment or the full-length protein was likely responsible for maintaining EEG abnormalities. Moreover, transgene suppression normalized the ratio of excitatory to inhibitory innervation in the cortex, whereas secretase inhibition did not. Our results suggest that APP overexpression, and not Aβ overproduction, is responsible for EEG abnormalities in our transgenic mice and can be rescued independently of pathology.
|Piericidin A aggravates Tau pathology in P301S transgenic mice.|
Höllerhage, M; Deck, R; De Andrade, A; Respondek, G; Xu, H; Rösler, TW; Salama, M; Carlsson, T; Yamada, ES; Gad El Hak, SA; Goedert, M; Oertel, WH; Höglinger, GU
PloS one 9 e113557 2014
The P301S mutation in exon 10 of the tau gene causes a hereditary tauopathy. While mitochondrial complex I inhibition has been linked to sporadic tauopathies. Piericidin A is a prototypical member of the group of the piericidins, a class of biologically active natural complex I inhibitors, isolated from streptomyces spp. with global distribution in marine and agricultural habitats. The aim of this study was to determine whether there is a pathogenic interaction of the environmental toxin piericidin A and the P301S mutation.Transgenic mice expressing human tau with the P301S-mutation (P301S+/+) and wild-type mice at 12 weeks of age were treated subcutaneously with vehicle (N = 10 P301S+/+, N = 7 wild-type) or piericidin A (N = 9 P301S+/+, N = 9 wild-type mice) at a dose of 0.5 mg/kg/d for a period of 28 days via osmotic minipumps. Tau pathology was measured by stereological counts of cells immunoreative with antibodies against phosphorylated tau (AD2, AT8, AT180, and AT100) and corresponding Western blot analysis.Piericidin A significantly increased the number of phospho-tau immunoreactive cells in the cerebral cortex in P301S+/+ mice, but only to a variable and mild extent in wild-type mice. Furthermore, piericidin A led to increased levels of pathologically phosphorylated tau only in P301S+/+ mice. While we observed no apparent cell loss in the frontal cortex, the synaptic density was reduced by piericidin A treatment in P301S+/+ mice.This study shows that exposure to piericidin A aggravates the course of genetically determined tau pathology, providing experimental support for the concept of gene-environment interaction in the etiology of tauopathies.
|DNA methylation impairs TLR9 induced Foxp3 expression by attenuating IRF-7 binding activity in fulminant type 1 diabetes.|
Wang, Zhen, et al.
J. Autoimmun., 41: 50-9 (2013) 2013
Fulminant type 1 diabetes (FT1D) is an extremely aggressive disease characterized by the abrupt onset of insulin-deficient hyperglycemia. However, the precise mechanisms underlying disease etiology almost remain unclear. As mice deficient in regulatory T cells (Tregs) are prone to the development of an FT1D-like phenotype, we thus investigated whether FT1D patients manifest Treg deficiency and explored the related mechanisms. We first noted a significant reduction for Foxp3 and CTLA4 expression levels in PBMCs of FT1D patients. IRF-7 was found to selectively bind to the Foxp3 promoter, and by which it promotes Foxp3 transcription. Therefore, ectopic IRF-7 expression significantly promoted Foxp3 and CTLA4 expression in PBMCs, while knockdown of IRF-7 manifested opposite effect. Importantly, stimulation of PBMCs with CpG ODN, a ligand for TLR9, significantly induced Foxp3 expression, demonstrating that TLR9 signaling positively regulates Treg development. However, knockdown of IRF-7 expression almost completely diminished the enhancing effect of TLR9 signaling on Foxp3 expression, suggesting that IRF-7 is a downstream molecule of TLR9 signaling and is essential for TLR9 induced Treg generation. Of interestingly note, the Foxp3 promoter in FT1D patients was hypermethylated, indicating that DNA methylation could be a causative factor responsible for the reduced Foxp3 expression in FT1D patients. Indeed, our mechanistic studies revealed that DNA methylation blocked IRF-7 binding to the Foxp3 promoter. Together, our data support the notion that environmental insults in genetic predisposed subjects trigger Foxp3 promoter hypermethylation, which then prevents IRF-7 binding to the Foxp3 promoter and impairs Treg development/functionality contributing to the pathogenesis of FT1D.
|Profiling murine tau with 0N, 1N and 2N isoform-specific antibodies in brain and peripheral organs reveals distinct subcellular localization, with the 1N isoform being enriched in the nucleus.|
Liu, C; Götz, J
PloS one 8 e84849 2013
In the adult murine brain, the microtubule-associated protein tau exists as three major isoforms, which have four microtubule-binding repeats (4R), with either no (0N), one (1N) or two (2N) amino-terminal inserts. The human brain expresses three additional isoforms with three microtubule-binding repeats (3R) each. However, little is known about the role of the amino-terminal inserts and how the 0N, 1N and 2N tau species differ. In order to investigate this, we generated a series of isoform-specific antibodies and performed a profiling by Western blotting and immunohistochemical analyses using wild-type mice in three age groups: two months, two weeks and postnatal day 0 (P0). This revealed that the brain is the only organ to express tau at significant levels, with 0N4R being the predominant isoform in the two month-old adult. Subcellular fractionation of the brain showed that the 1N isoform is over-represented in the soluble nuclear fraction. This is in agreement with the immunohistochemical analysis as the 1N isoform strongly localizes to the neuronal nucleus, although it is also found in cell bodies and dendrites, but not axons. The 0N isoform is mainly found in cell bodies and axons, whereas nuclei and dendrites are only slightly stained with the 0N antibody. The 2N isoform is highly expressed in axons and in cell bodies, with a detectable expression in dendrites and a very slight expression in nuclei. The 2N isoform that was undetectable at P0, in adult brain was mainly found localized to cell bodies and dendrites. Together these findings reveal significant differences between the three murine tau isoforms that are likely to reflect different neuronal functions.