Fig4G_RACE_STOML2_R1_LG349.sgd

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蛋白或空载体，以及携带STOML2来源15 bp插入序列的荧光素酶报告基因；该类报告基因包含三种类型：野生型、GC碱基对发生突变以破坏发夹结构的突变体，以及GC碱基对发生突变但保留发夹结构的突变体。24小时后收集细胞RNA，采用结合荧光素酶序列的引物，通过5' 快速扩增cDNA末端（5' RACE）进行分析。