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  • PI3K is negatively regulated by PIK3IP1, a novel p110 interacting protein. 17475214

    Signaling initiated by Class Ia phosphatidylinositol-3-kinases (PI3Ks) is essential for cell proliferation and survival. We discovered a novel protein we call PI3K interacting protein 1 (PIK3IP1) that shares homology with the p85 regulatory PI3K subunit. Using a variety of in vitro and cell based assays, we demonstrate that PIK3IP1 directly binds to the p110 catalytic subunit and down modulates PI3K activity. Our studies suggest that PIK3IP1 is a new type of PI3K regulator.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-195

  • Development of multiple cell-based assays for the detection of histone H3 Lys27 trimethylation (H3K27me3). 23992119

    Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA(®) and LanthaScreen(®) technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA(®)) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds.
    Tipo de documento:
    Referencia
    Referencia del producto:
    ABE44
    Nombre del producto:
    Anti-trimethyl Histone H3 (Lys27) Antibody

  • Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration. 20203200

    Aggregation of alpha-synuclein (alpha-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric alpha-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we used two-dimensional analytical separation and an array of biochemical and cell-based assays to characterize alpha-syn oligomers that are present in the nervous system of A53T alpha-syn transgenic mice. The most prominent species identified were 53 A detergent-soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing alpha-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea but were sensitive to proteinase-K digestion. Although the oligomers shared similar basic biochemical properties, those obtained from inclusion-bearing regions were prominently reactive to antibodies that recognize oxidized alpha-syn oligomers, significantly accelerated aggregation of alpha-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion-bearing regions were not toxic and delayed the in vitro formation of alpha-syn fibrils. These data indicate that specific conformations of alpha-syn oligomers are present in distinct brain regions of A53T alpha-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region-specific intrinsic factors.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB369
    Nombre del producto:
    Anti-Dopamine Transporter Antibody, NT, clone DAT-Nt

  • Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling. 25246557

    The mitotic checkpoint (also known as the spindle assembly checkpoint) prevents premature anaphase onset through generation of an inhibitor of the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. Combining in vitro reconstitution and cell-based assays, we now identify dual mechanisms through which Bub3 promotes mitotic checkpoint signaling. Bub3 enhances signaling at unattached kinetochores not only by facilitating binding of BubR1 but also by enhancing Cdc20 recruitment to kinetochores mediated by BubR1's internal Cdc20 binding site. Downstream of kinetochore-produced complexes, Bub3 promotes binding of BubR1's conserved, amino terminal Cdc20 binding domain to a site in Cdc20 that becomes exposed by initial Mad2 binding. This latter Bub3-stimulated event generates the final mitotic checkpoint complex of Bub3-BubR1-Cdc20 that selectively inhibits ubiquitination of securin and cyclin B by APC/C(Cdc20). Thus, Bub3 promotes two distinct BubR1-Cdc20 interactions, involving each of the two Cdc20 binding sites of BubR1 and acting at unattached kinetochores or cytoplasmically, respectively, to facilitate production of the mitotic checkpoint inhibitor.
    Tipo de documento:
    Referencia
    Referencia del producto:
    16-213
    Nombre del producto:
    Anti-Myc Tag Antibody, clone 4A6, HRP conjugate

  • Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo. 17942691

    The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Small molecule R1498 as a well-tolerated and orally active kinase inhibitor for hepatocellular carcinoma and gastric cancer treatment via targeting angiogenesis and mitos ... 23755206

    Protein kinases play important roles in tumor development and progression. Lots of kinase inhibitors have entered into market and show promising clinical benefits. Here we report the discovery of a novel small molecule, well-tolerated, orally active kinase inhibitor, R1498, majorly targeting both angiogenic and mitotic pathways for the treatment of hepatocellular carcinoma (HCC) and gastric cancer (GC). A series of biochemical and cell-based assays indicated that the target kinase cluster of R1498 included Aurora kinases and VEGFR2 et al. R1498 showed moderate in vitro growth inhibition on a panel of tumor cells with IC50 of micromole range. The in vivo anti-tumor efficacy of R1498 was evaluated on a panel of GC and HCC xenografts in a parallel comparison with another multikinase inhibitor sorafenib. R1498 demonstrated superior efficacy and toxicity profile over sorafenib in all test models with greater than 80% tumor growth inhibition and tumor regression in some xenogratfts. The therapeutic potential of R1498 was also highlighted by its efficacy on three human GC primary tumor derived xenograft models with 10-30% tumor regression rate. R1498 was shown to actively inhibit the Aurora A activity in vivo, and decrease the vascularization in tumors. Furthermore, R1498 presented good in vivo exposure and therapeutic window in the pharmacokinetic and dose range finding studies. Theses evidences indicate that R1498 is a potent, well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic and antiproliferative profile, and provide strong confidence for further development for HCC and GC therapy.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-368
    Nombre del producto:
    Anti-phospho-Ser/Thr-Pro MPM-2 Antibody

  • Differential regulation of Gli proteins by Sufu in the lung affects PDGF signaling and myofibroblast development. 24886827

    Mammalian Hedgehog (Hh) signaling relies on three Gli transcription factors to mediate Hh responses. This process is controlled in part by a major negative regulator, Sufu, through its effects on Gli protein level, distribution and activity. In this report, we showed that Sufu regulates Gli1 protein levels by antagonizing Numb/Itch. Otherwise, Numb/Itch would induce Gli1 protein degradation. This is in contrast to inhibition of Spop-mediated degradation of Gli2/3 by Sufu. Thus, controlling protein levels of all three Gli genes by Sufu is a conserved mechanism to modulate Hh responses albeit via distinct pathways. These findings in cell-based assays were further validated in vivo. In analyzing how Sufu controls Gli proteins in different tissues, we discovered that loss of Sufu in the lung exerts different effects on Hh target genes. Hh targets Ptch1/Hhip are upregulated in Sufu-deficient lungs, consistent with Hh pathway activation. Surprisingly, protein levels of Hh target Gli1 are reduced. We also found that myofibroblasts are absent from many prospective alveoli of Sufu-deficient lungs. Myofibroblast development is dependent on PDGF signaling. Interestingly, analysis of the Pdgfra promoter revealed a canonical Gli-binding site where Gli1 resides. These studies support a model in which loss of Sufu contributes to compromised Pdgfra activation and disrupts myofibroblast development in the lung. Our work illustrates the unappreciated complexity of Hh responses where distinct Hh targets could respond differently depending on the availability of Gli proteins that control their expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • Establishment of a cell-based assay for screening of compounds inhibiting very early events in the cytomegalovirus replication cycle and characterization of a compound id ... 18458124

    To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 microM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB810