|
Author:
Date:
2013-10-20
[Abstract] Fluorescence Recovery After Photobleaching (FRAP) (Lippincott-Schwartz et al., 2003; Reits and Neefjes, 2001) was employed to determine dynamic properties of proteins localized at the ephitelial zonula adherens (ZA) (Kovacs et al., 2011; Otani et al., 2006). The proteins of interest were expressed in cells using a knockdown and reconstitution approach in which endogenous proteins were depleted by RNA interference (RNAi) and replaced by expression of an RNAi-resistant gene fused to GFP (Priya et al., 2013; Smutny et al., 2010; Smutny et al., 2011; Vitriol et al., 2007). By choosing expression levels of GFP-tagged proteins that were comparable to endogenous levels, we minimized transient overexpression artifacts due to ...
[摘要] 使用光漂白后的荧光恢复（FRAP）（Lippincott-Schwartz等人，2003; Reits和Neefjes，2001）来确定位于Ephitelial zonula adherens（ZA）处的蛋白质的动力学性质（Kovacs < et="" al。，2011;="" otani="" et="" al。，2006）。使用敲除和重建方法在细胞中表达感兴趣的蛋白质，其中内源蛋白质被rna干扰（rnai）耗尽，并被与gfp融合的rnai抗性基因的表达代替（priya等人。="">，2013; Smutny等人，2010; Smutny等人，2011; Vitriol等人，2007）。通过选择与内源水平相当的GFP标记蛋白的表达水平，我们最小化了由于克服了直接影响蛋白质动力学的调节机制的瞬时过表达人工产物（Goodson等人，2010）。使用这种方法，使用共聚焦显微镜检查，在对应于ZA的小区域中对连接的E-钙粘蛋白-GFP或GFP-Ect2进行FRAP分析（Priya等人，2013; Ratheesh等人。，2012; Gomez等人，2005; ...
|