CIL:25702

MTLn3 cell cotransfected with FAK siRNA (target A) and two tandem Src SH2 phosphotyrosine-binding domains (YFP-dSH2), which specifically detects tyrosine phosphorylation at focal adhesions. CIL:25703 is the corresponding control experiment. Fluorescence imaging was performed using a 60×/1.40 oil objective on an inverted microscope (1X-70;Olympus) in a 37°C closed system as previously described. Glass-bottomed dishes (35 mm) were coated with 10 µg/ml fibronectin for 1 h at 37°C. Cells were plated in Ham’s F12 containing 5% FBS and 20 mM Hepes, pH 7.2, and were allowed to adhere for 3 h. Fluorescent images were collected using a cooled CCD camera (CoolSNAP FX; Hamamatsu Photonics) and captured into MetaVue every 1 min for 1 h. Time-lapse sequences from live fluorescence imaging were first subjected to high-pass filtration based on the water algorithm (Zamir et al., 1999) to remove diffuse background fluorescence. Movie corresponds to video 5 from J Cell Biol. 2009 Apr 20;185(2):357-70. Epub 2009 Apr 13.

MTLn3细胞共转染了FAK小干扰RNA（FAK siRNA，靶标A）以及两个串联的Src SH2磷酸酪氨酸结合结构域（YFP-dSH2），该探针可特异性检测黏着斑处的酪氨酸磷酸化。CIL:25703为对应的对照实验。荧光成像采用60×/1.40油浸物镜搭配倒置显微镜（1X-70；奥林巴斯（Olympus））完成，实验在37℃密闭系统中进行，操作流程参照此前已发表的方法。35mm玻璃底培养皿经10μg/ml纤连蛋白于37℃下包被1小时。细胞接种于含5%胎牛血清（FBS）、20mM HEPES且pH值为7.2的Ham’s F12培养基中，贴壁培养3小时。使用制冷型CCD相机（CoolSNAP FX；滨松光子（Hamamatsu Photonics））采集荧光图像，以每分钟1帧的速率连续拍摄1小时，并将图像保存至MetaVue软件中。活细胞荧光延时成像的时序序列首先基于水算法（Zamir等，1999）进行高通滤波，以去除弥散的背景荧光。该影片对应《细胞生物学杂志（Journal of Cell Biology）》2009年4月20日刊第185卷第2期第357至370页的视频5，该文章于2009年4月13日在线发表。