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 × 106±7.86 × 104)、人宫颈癌细胞HeLa(6.59 ×106±1.79×106)和人肝癌细胞 HepG2(3.13 × 106±2.87 × 105)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=0.825 8)和 H1299(R=0.906 2),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 × 106±7.86 × 104),human cervical cancer cell HeLa(6.59 × 106±1.79 × 106)and human liver cancer cell HepG2(3.13 × 106±2.87 × 105)all changed significantly(all P＜0.05).The correlation results showed that for human non-small cell lung cancer cells A549(R=0.825 8)and H1299(R=0.906 2),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.
孙浚源;罗微微;杨金凤;贾静
杭州医学院检验医学与生物工程学院,浙江杭州 310013杭州医学院药学院,浙江杭州 310013
药学
内参蛋白;β-肌动蛋白;甘油醛-3-磷酸脱氢酶;细胞S期激酶相关蛋白2;相对表达水平;蛋白质印迹
internal reference protein;β-actin;glyceraldehyde-3-phosphate dehydrogenase;S-phase kinase-associated protein 2;relative expression level;Western Blot
《中国临床药理学杂志》 2024 (006)
844-848 / 5
国家自然科学基金资助项目(81802622);浙江省自然科学基金资助项目(LY18H160067)
10.13699/j.cnki.1001-6821.2024.06.013
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