HEK293和HeLa细胞分别被Np95/ICBP90-like RING finger protein (NIRF) 和P53转染后，细胞上清和免疫沉淀产物用SDS-聚丙烯酰胺凝胶电泳免疫印迹法分析；细菌合成GST-P53后，用GST pull-down技术检测NIRF与P53相互作用；在GST-P53、E1、E2和NIRF体外泛素化反应系统中，检测NIRF对P53的体外泛素化. 结果表明：NIRF能与P53相互作用，NIRF不仅能与P53特异性结合，而且还会将P53泛素化，这种相互作用在细胞内和细胞外均能发生. 推测NIRF可能是P53的一个新的负调节蛋白.
Abstract:
HEK293 or HeLa cells were transfected by NIRF and, or P53, whole cell extracts and immunoprecipitates were subjected to SDS-PAGE followed by Western blotting. GST pull-down was carried out to identify the interactions between NIRF and P53. In vitro ubiquitination reaction was carried out to identify P53 ubquitinate by NIRF. The results suggested that NIRF could interact with P53 in vivo and in vitro. The results also showed that NIRF could ubiquitinate P53 in vivo and in vitro. The results indicated that NIRF would be a new negative regulator of P53.
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