| Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
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Author:
Date:
2017-11-05
[Abstract] Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
[摘要] 通过研究其配体组织蛋白酶D的加工和分泌的改变，可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定，并且包括新合成的蛋白质的代谢标记，其中[35 [S]甲硫氨酸半胱氨酸，以监测组织蛋白酶D处理，通过分泌途径和分泌使用免疫沉淀，SDS-PAGE和荧光。
【背景】组织蛋白酶d（CATD）是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体（M6PRs）排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体（戈什等人，2003 ）。将CatD合成为前体蛋白（〜52kDa），其在溶酶体中被切割以产生中间体（〜48kDa）或成熟的溶酶体形式（〜34kDa）。微量的前体蛋白质也从生物合成途径分泌（Benes et al。，2008）。 catD的丰度可以使用几种方法来确定，例如基于免疫荧光的染色（Poole等人，1972），蛋白质印迹，荧光活性测定（Bewley等人， （Hirst等人，2009; Kametaka等人，2007; Tavares等人，2011）或用脉冲追踪分析进行代谢标记（Hirst等人，2009; Kametaka等人， ...
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| VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells
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Author:
Date:
2017-02-20
[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the ...
[摘要] 造血干细胞（HSC）由其自我更新的功能能力定义，并分化成所有血细胞谱系。大多数HSC位于骨髓（BM）微环境中的特定解剖位置，处于静止非运动模式。 HSCs与其支持的BM微环境细胞之间的粘附相互作用对于维持干细胞静止和保护免受DNA损伤因子阻止血液学失败和死亡至关重要。多重信号蛋白在控制骨髓HSCs的保留和迁移中起重要作用。粘附分子参与调节造血和干细胞和祖细胞BM保留，迁移和发育的两个过程。干细胞从骨髓移动到骨髓的机制尚未完全阐明，仍然是深入研究的对象。一个重要的方面是修饰干细胞保留，迁移和发育所需的干细胞和祖细胞的粘附分子的表达和亲和力。粘附力通过粘附分子的表达，亲和力和亲合力来调节。亲和力调节与分子结合识别和结合强度有关。在这里，我们描述了在我们的研究中使用的体外 FACS测定来研究整联蛋白α4亚类β1亚单位的表达，亲和力和功能也称为VLA-4）用于小鼠骨髓保留EPCR +长期复制HSC（LT-HSC）（Gur-Cohen等人，2015）。背景整合素是介导细胞和细胞 - ...
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| Sample Preparation for X-ray Micro-computed Tomography of Woody Plant Material and Associated Xylem Visualisation Techniques
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Author:
Date:
2016-03-20
[Abstract] Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow ...
[摘要] 短旋转枝（SRC）柳树的组织结构的变化是驱动木质纤维素糖产量的差异的主要因素，但是该组织的大部分生理学和发育是未知的。传统的切片在木质组织中既困难又具有破坏性;然而，诸如三维X射线微计算断层扫描（μCT）扫描的技术可以用于移动生物学研究者超越组织变化的传统二维评估，而不必破坏性地切割细胞。这种技术不能取代传统的微观技术，而是可以仔细地与传统方法整合，以改进植物生物学世界的三维探索。以下程序概述了用于3D X射线μCT和相关木质部染色和可视化技术的柳树的制备，特别是在凝胶状纤维（g-纤维）发育期间的次生木质部程序性细胞死亡（PCD）延迟。这里的许多染色技术可转移到其他木本物种，如杨树和桉树。
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