Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13700, Chlorocebus aethiops. CIL. Dataset

This is one of a group of four images in Figure S5 in Apaja et al., JCB 2010, that support the conclusion that the WT vasopressin type2 receptor (V2R) and the V2R W164S mutant (that causes nephrogenic diabetes insipidus) have different post-endocytic targeting. The sorting of endocytosed myc-tagged WT and W164S V2R was determined by indirect immunolabeling of internalized receptors in transiently transfected COS7 cells. The WT and temperature (26 degrees C) rescued mutant receptors were labelled with anti-myc Ab in live cells (1h) and then chased in Ab-free medium (1h, 37 degrees C) before indirect immunolabeling. To mark lysosomes, cells were labeled with 10 kD Texas red–dextran at 50 µg/ml by overnight fluid-phase endocytosis and chased for >3 h at 37°C. Fluorescence micrographs were obtained by a microscope (LSM510 or LSM710; Carl Zeiss, Inc.) equipped with a Plan-Apochromat 63×/NA 1.4 objective in multitrack mode. This single optical section shows that the WT V2R does not co-localize with lysosomes. A companion image shows that the internalized W164S V2R mutant, however, does co-localize with lysosomes.

此描述为Apaja等人在2010年发表在《JCB》期刊的图S5中的四幅图像组之一，旨在支持以下结论：野生型（WT）血管加压素受体2型（V2R）与V2R W164S突变型（引发肾性尿崩症）在胞内转运方面存在差异。通过间接免疫标记法对瞬时转染COS7细胞内吞的带有myc标记的WT和W164S V2R的排序进行了确定。在活细胞中，野生型和温度（26摄氏度）恢复的突变型受体用抗-myc抗体标记（1小时），然后在无抗体的培养基中追踪（1小时，37摄氏度）之后进行间接免疫标记。为了标记溶酶体，细胞用10 kD的Texas red-葡萄糖胺进行标记，浓度为50 µg/ml，通过过夜流体相内吞，并在37°C下追踪超过3小时。荧光显微镜（LSM510或LSM710；卡尔·蔡司公司）在多通道模式下，配备有Plan-Apochromat 63×/NA 1.4物镜，获取了荧光显微照片。单一光学切片显示野生型V2R与溶酶体不共定位。伴随的图像则显示，内化的W164S V2R突变型，然而，与溶酶体共定位。