Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.
Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.
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DOI：
10.1016/j.jim.2015.03.018
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发表时间：
2015-08
影响因子：
2.2
通讯作者：
Minderman H
中科院分区：
文献类型：
--
作者：
Maguire O;O'Loughlin K;Minderman H
核因子-κ B（NF-κB）的异常活性与许多疾病相关，并且是治疗靶点。翻译后修饰，特别是RELA/p65亚基的磷酸化，对于NF-κB/p65的胞质至核定位和下游靶基因转录的启动是必不可少的。免疫印迹和磷酸化流式细胞术已被用于研究磷酸化基序与NF-κB活化之间的关系，并且p65的核定位的显微镜分析也被用作活化的参数。这些方法的劳动密集性质通常限制了采样点或重复的数量。最近对p65磷酸化基序与其核定位之间关系的研究表明，这些参数具有不同的意义，不应互换使用。在这项研究中，我们证明的可行性和重复性研究之间的关系p65磷酸化和核转位成像流式细胞术（IFC）。在细胞系模型和健康供体淋巴细胞中，TNFα或PMA/离子霉素诱导的p65丝氨酸529磷酸化作为实验模型。IFC分析表明，磷酸化丝氨酸529（P-p65 s529）的表达迅速增加刺激后，P-p65 s529的核定位后，总p65的核定位模式。然而，在他克莫司的存在下，P-p65 s529的表达被抑制，而不影响总p65的核定位。这些数据表明，IFC的应用，同时评估磷酸化的p65和它的细胞定位和通过这种分析所获得的结果证实了目前的见解，他克莫司对丝氨酸529磷酸化的具体影响。
Aberrant activity of Nuclear Factor–kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry has been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights in the relationship between p65 phosphorylation motifs and its nuclear localization indicate that these parameters have different significance and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65s529) increased rapidly following stimulation and that nuclear localization of P-p65s529 followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65s529 expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.