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  • c-Fos protein expression is increased in cholinergic neurons of the rodent basal forebrain during spontaneous and induced wakefulness. 19716862

    It has been proposed that cholinergic neurons of the basal forebrain (BF) may play a role in vigilance state control. Since not all vigilance states have been studied, we evaluated cholinergic neuronal activation levels across spontaneously occurring states of vigilance, as well as during sleep deprivation and recovery sleep following sleep deprivation. Sleep deprivation was performed for 2h at the beginning of the light (inactive) period, by means of gentle sensory stimulation. In the rodent BF, we used immunohistochemical detection of the c-Fos protein as a marker for activation, combined with labeling for choline acetyl-transferase (ChAT) as a marker for cholinergic neurons. We found c-Fos activation in BF cholinergic neurons was highest in the group undergoing sleep deprivation (12.9% of cholinergic neurons), while the spontaneous wakefulness group showed a significant increase (9.2%), compared to labeling in the spontaneous sleep group (1.8%) and a sleep deprivation recovery group (0.8%). A subpopulation of cholinergic neurons expressed c-Fos during spontaneous wakefulness, when possible confounds of the sleep deprivation procedure were minimized (e.g., stress and sensory stimulation). Double-labeling in the sleep deprivation treatment group was significantly elevated in select subnuclei of the BF (medial septum/vertical limb of the diagonal band, horizontal limb of the diagonal band, and the magnocellular preoptic nucleus), when compared to spontaneous wakefulness. These findings support and provide additional confirming data of previous reports that cholinergic neurons of BF play a role in vigilance state regulation by promoting wakefulness.
    Document Type:
    Reference
    Product Catalog Number:
    AB143
    Product Catalog Name:
    Anti-Choline Acetyltransferase (ChAT) Antibody

  • Prion protein oligomers in Creutzfeldt-Jakob disease detected by gel-filtration centrifuge columns. 19389076

    Prion diseases are diagnosed by the detection of accumulation of abnormal prion protein (PrP) using immunohistochemistry or the detection of protease-resistant abnormal PrP (PrP(res)). Although the abnormal PrP is neurotoxic by forming aggregates, recent studies suggest that the most infectious units are smaller than the amyloid fibrils. In the present study, we developed a simplified method by applying size-exclusion gel-filtration chromatography to examine PrP oligomers without proteinase K digestion in Creutzfeldt-Jakob disease (CJD) samples, and evaluated the correlation between disease severity and the polymerization degree of PrP. Brain homogenates of human CJD and non-CJD cases were applied to the gel-filtration spin columns, and fractionated PrP molecules in each fraction were detected by western blot. We observed that PrP oligomers could be detected by the simple gel-filtration method and distinctly separated from monomeric cellular PrP (PrP(c)). PrP oligomers were increased according to the disease severity, accompanied by the depletion of PrP(c). The separated PrP oligomers were already protease-resistant in the case with short disease duration. In the cases with quite severe pathology the oligomeric PrP reached a plateau, which may indicate that PrP molecules could mostly develop into amyloid fibrils in the advanced stages. The increase of PrP oligomers correlated with the degree of histopathological changes such as spongiosis and gliosis. The decrease of monomeric PrP(c) was unexpectedly obvious in the diseased cases. Dynamic changes of both oligomerization of the human PrP and depletion of normal PrP(c) require further elucidation to develop a greater understanding of the pathogenesis of human prion diseases.
    Document Type:
    Reference
    Product Catalog Number:
    AP192P
    Product Catalog Name:
    Donkey Anti-Mouse IgG Antibody, HRP conjugate, Species Adsorbed

  • A protein profile study to discriminate CIN lesions from normal cervical epithelium. 21573931

    Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. 24023619

    Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.
    Document Type:
    Reference
    Product Catalog Number:
    AB9568
    Product Catalog Name:
    Anti-Neurofilament L Antibody

  • Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption. 23401167

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.
    Document Type:
    Reference
    Product Catalog Number:
    05-321
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B ... 18854601

    The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.
    Document Type:
    Reference
    Product Catalog Number:
    S7110
    Product Catalog Name:
    ApopTag® Fluorescein In Situ Apoptosis Detection Kit

  • A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity. 11399042

    We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.
    Document Type:
    Reference
    Product Catalog Number:
    05-429
    Product Catalog Name:
    Anti-phospho-MBP Antibody, clone P12