Chang Ji Suk, Gettys Thomas W
Laboratory of Nutrient Sensing and Adipocyte Signaling, Pennington Biomedical Research Center, Baton Rouge, LA, USA.
Methods Mol Biol. 2013;952:163-73. doi: 10.1007/978-1-62703-155-4_11.
Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates the genes involved in energy metabolism. Recent evidence has been provided that alternative splicing of PPARGC1A gene produces a functional but predominantly cytosolic isoform of PGC-1α (NT-PGC-1α). We have demonstrated that transcriptional coactivation capacity of NT-PGC-1α is directly correlated with its nuclear localization in a PKA phosphorylation-dependent manner. In this chapter, we describe quantitative imaging analysis methods that are developed to measure the relative fluorescence intensity of the protein of interest in the nucleus and cytoplasm in a single cell and the frequency distribution of nuclear/cytoplasmic intensity ratios in the population of cells, respectively. This chapter also describes transient cotransfection and dual-luciferase reporter gene assay that examine the ability of coactivators to activate the transcriptional activity of transcription factors.
过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）是一种核转录共激活因子，可调节参与能量代谢的基因。最近有证据表明，PPARGC1A基因的可变剪接产生了一种功能性但主要位于胞质的PGC-1α异构体（NT-PGC-1α）。我们已经证明，NT-PGC-1α的转录共激活能力与其以PKA磷酸化依赖方式的核定位直接相关。在本章中，我们描述了所开发的定量成像分析方法，分别用于测量单个细胞中细胞核和细胞质中目标蛋白的相对荧光强度以及细胞群体中核/质强度比的频率分布。本章还描述了瞬时共转染和双荧光素酶报告基因测定，用于检测共激活因子激活转录因子转录活性的能力。
