用蛋白质印迹评价GAPDH与β-actin作为Skp2内参蛋白的研究

Evaluation of GAPDH and β-actin as internal reference proteins of Skp2 by Western blot

目的确定用蛋白质印迹(WB)技术研究细胞S期激酶相关蛋白2(Skp2)时适宜的内参蛋白。方法选取多种人源肿瘤细胞系,通过瞬时转染shRNA质粒下调细胞内Skp2蛋白水平,用甘油醛-3-磷酸脱氢酶(GAPDH)与β-肌动蛋白(β-actin)作为内参蛋白进行WB检测,分析GAPDH、β-actin、Skp2的相对表达水平及相互关系。结果细胞内Skp2相对表达水平发生改变时,对于人非小细胞肺癌细胞H1299(1.32×10^(6)±7.86×10^(4))、人宫颈癌细胞HeLa(6.59×10^(6)±1.79×10^(6))和人肝癌细胞HepG2(3.13×10^(6)±2.87×10^(5))的GAPDH相对表达水平均发生明显变化(均P<0.05)。相关性结果显示:对于人非小细胞肺癌细胞A549(R=0.8258)和H1299(R=0.9062),Skp2与GAPDH相对表达水平均存在一定的相关性(均P<0.05)。结论β-actin更适合作为研究Skp2蛋白WB实验的内参蛋白。

Objective To determine the appropriate internal reference protein for the Western Blot(WB)assay to detect proteins related to S-phase kinase-associated protein 2(Skp2).Methods This study was carried out with several human tumor cell lines.The intracellular Skp2 protein level was down-regulated by transient transfection of shRNA plasmid,and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and β-actin were used as internal reference proteins respectively for WB assay.The relative expression levels and correlations of GAPDH,β-actin and Skp2 were evaluated.Results When the relative expression level of Skp2 changed,the relative expression levels of GAPDH in human non-small cell lung cancer cells H1299(1.32×10^(6)±7.86×10^(4)),human cervical cancer cell HeLa(6.59×10^(6)±1.79×10^(6))and human liver cancer cell HepG2(3.13×10^(6)±2.87×10^(5))all changed significantly(all P<0.05).The correlation results showed that for human non-small cell lung cancer cells A549(R=0.8258)and H1299(R=0.9062),and there was a certain correlation between Skp2 and GAPDH expression levels(all P<0.05).Conclusion β-actin is more suitable to be applied as reference protein in WB assay for Skp2 protein.