Fig4G_RACE_STOML2_R2_LG350_annotated.tif

HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with PA-X from the influenza A/Puerto Rico/8/1984 (H1N1) or vector, and luciferase reporters with a 15 bp inserted sequences from the STOMl2, wild-type or containing mutations in GC basepairs to disrupt the hairpin structure, or contanins mutations in GC basepairs that preserve the hairpin structure. RNA was collected 24 hrs later and analyze by 5' RACE using a primer that binds to luciferase sequences.

将HEK293T ishXrn1细胞经多西环素处理3至4天以诱导Xrn1基因敲低，随后分别转染甲型流感病毒A/波多黎各/8/1984（H1N1）亚型来源的PA-X蛋白表达质粒或空载体，同时转染携带三类15 bp插入序列的荧光素酶报告基因载体：插入序列均取自STOMl2，分别为野生型序列、经GC碱基对突变以破坏发卡结构的突变序列，以及经GC碱基对突变但保留发卡结构的突变序列。于转染后24小时收集RNA，采用结合荧光素酶序列的引物进行5' 末端快速扩增（5' Rapid Amplification of cDNA Ends, 5' RACE）分析。