| Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
|
|
Author:
Date:
2020-07-20
[Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of ...
[摘要] [摘要 t] 紧密连接蛋白claudin家族的成员调节细胞旁通透性并调节细胞信号传导。在连接重塑过程中，这些蛋白被选择性地插入紧密连接或从紧密连接中检索出来，但是对这些过程的控制和协调仍不完全了解。claudins的可视化可以评估其定位和丰度的变化。我们使用所描述的方案对claudin-2染色，但它也可以用于染色任何紧密连接蛋白。我们发现使用甲醇进行固定可以使claudin-2在膜和细胞质囊泡中得到最佳保存。使用claudin-2特异性一抗和荧光标记的二抗以及与DAPI标记核一起进行染色。然后使用共聚焦显微镜对样品成像，并获得z堆栈，从而可以可视化连接和细胞内claudin-2。总的claudin-2信号可以在3D重建图像后使用Imaris 软件。
[背景 ] 紧密连接（TJ）是一种多蛋白复合物，位于连接上皮细胞的细胞间连接复合物的最顶端（Van Itallie和Anderson，2014）。这些结构产生通透性屏障和离子特异性旁细胞途径，维持apicobasal 极性，为各种重要功能提供输入并调节信号传导途径。位于TJs的蛋白可分为跨膜蛋白和相关的胞质蛋白（综述见（González-Mariscal ...
|
|
|
| Cell-based Assays to Monitor AID Activity
|
|
Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which ...
[摘要] 酶活化诱导的脱氨酶（AID）通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变（SHM）和类型转换重组（CSR）的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1）AID突变大肠杆菌基因组的能力，其提供其催化活性的指示; 2）AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3）AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合，伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照，允许AID的详细结构功能研究。
|
|
|