Fig4G_RACE_STOML2_R2_LG350.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.

将诱导型敲低Xrn1的HEK293T细胞采用多西环素处理3~4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）来源的PA-X蛋白编码序列或空载体，同时转染携带不同15bp插入序列的荧光素酶报告基因：插入序列均来自STOMl2基因，包括野生型序列、突变GC碱基对以破坏发夹结构的突变序列，以及突变GC碱基对但保留发夹结构的突变序列。转染24小时后收集RNA，使用结合荧光素酶序列的引物通过5'末端快速扩增（5' RACE, rapid amplification of cDNA ends）技术进行分析。