Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER)
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Author:
Date:
2017-04-05
[Abstract] In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized ...
[摘要] 在该方案中,我们描述了一种定量生化测定法来评估内质网(ER)对脂肪细胞中高尔基体蛋白质转运的效率(Bruno等,2016)。 该测定法利用脂肪细胞分泌各种生物活性蛋白质(称为脂肪因子)的事实。 作为对高尔基体通量的ER的测量,我们使用免疫印迹法确定了使用Brefeldin A(BFA)处理的脂肪因子adipsin后消失的体积分泌速率。 因为BFA治疗导致ER中Adipsin的积累,所以在BFA洗脱时,Adipsin从ER的出口在细胞和实验条件下同步。 因此,使用这种简单的测定法可以稳健地确定扰动,如敲低蛋白质,是否对高尔基蛋白转运的ER有影响。 【背景】新合成的蛋白质通过分泌途径从细胞分泌到细胞膜(PM)中。分泌路线包括从ER到高尔基体的运输,穿过高尔基体堆叠,以及从高尔基网络(TGN)到PM的运动。这些运输步骤中的每一个提供用于调节分泌物的节点。虽然大多数细胞能够分泌蛋白质,但某些特定的细胞类型是特异性蛋白质的专业秘密。脂肪细胞,例如,分泌激素,称为脂肪因子,影响各种器官的能量代谢。为了更好地了解脂肪因子分泌的分子基础,我们开发了一种测定方法,研究了从ER到脂肪细胞的高尔基体的脂肪因子,一种脂肪因子的转运。研究ER到高尔基体运输的传统和常用的方法是监测从ER融合到GFP(VSVG-GFP)的温度敏感性水泡性口炎病毒G蛋白ts045的出口(Presley等,1997)。使用adipsin分泌测定法研究脂肪细胞中的高铁运输的优点是监测来自ER的内源性蛋白质的通量,而不是异位表达的报道蛋白。 ...
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Cell Wall-bound p-Coumaric and Ferulic Acid Analysis
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Author:
Date:
2016-10-05
[Abstract] Hydroxycinnamic acids, such as p-coumaric acid and ferulic acid, are a major class of compounds derived from the phenylpropanoid pathway. These compounds are widely conserved in plants and primarily accumulate in the secondary cell wall. They serve as important structural components that contribute to the overall strength and rigidity of plant cell walls and are also potent antioxidants valued for nutritional consumption. This protocol describes a method for analyzing hydroxycinnamic acids that are released after incubation under alkaline conditions.
[摘要] 羟基肉桂酸例如对 - 香豆酸和阿魏酸是衍生自苯丙素途径的一类主要化合物。 这些化合物在植物中广泛保守,并且主要累积在继发性细胞壁中。 它们作为重要的结构组分,有助于植物细胞壁的整体强度和刚性,并且是有价值的营养消耗的有效抗氧化剂。 该方案描述了用于分析在碱性条件下孵育后释放的羟基肉桂酸的方法。
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Quantification of Rae-1 Staining Intensity in Glomeruli
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Author:
Date:
2016-03-05
[Abstract] NKG2D is expressed on all NK cells and on subsets of NKT, CD8, CD4 and γδ T cells. NKG2D is activated by NKG2D ligands, a stress-induced family of MHC-I-like proteins. These ligands are upregulated on stressed/infected cells and are not widely expressed on healthy adult tissue. NKG2D ligands have been widely studied as potential targets for immunotherapeutic approaches in cancer and auto-immune diseases such as Systemic Lupus Erythematosus. Here we describe a method for quantifying the expression levels of the Rae-1 NKG2D ligand in the glomeruli of healthy and diseased individuals via a novel ...
[摘要] NKG2D在所有NK细胞上和在NKT,CD8,CD4和γδT细胞的子集上表达。 NKG2D被NKG2D配体激活,NKG2D配体是应激诱导的MHC-I样蛋白家族。这些配体在应激/感染的细胞上上调,并且不在健康成人组织上广泛表达。 NKG2D配体已被广泛研究作为在癌症和自身免疫疾病如系统性红斑狼疮中的免疫治疗方法的潜在靶标。在这里我们介绍一种方法通过一个新的算法,量化健康和患病个体的肾小球Rae-1 NKG2D配体的表达水平。 MRL小鼠用作阳性对照菌株,因为其自发产生狼疮样表型,其中一个主要影响是严重的肾小球肾炎。 MRL/MpJ小鼠在约12个月龄时自发产生该表型,而具有相同遗传背景但在Fas等位基因中产生自发纯合突变的MRL/MpJlpr小鼠发展类似但更严重的症状〜3 -4个月龄(Spada等人,2015)。
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