Figure2 .

PTBP1 promotes IRES-mediated translation of cyclin B1

(A) The RNA bands of cyclin B1 and c-myc 5′UTR. RNA sequence of cyclin B1 and c-myc 5′UTR were transcriptions from DNA template using the MEGAscript® kits. CTP and UTP were labeled with biotin. (B) RNA pull-down assay of cyclin B1 5′UTR. The RNA sequences were synthesized by in vitro transcription. Biotin UTPs and CTPs were added to the reaction. The MYC 5′UTR sequence was used as a positive control and the HAV 617-740 sequence as a negative control. (C) RNA IP assay of PTBP1. The experiment was performed by using the Magna RIPTM RNA-Binding Protein Immunoprecipitation kit. The cell lysate was from KYSE 30 cells. Data are shown as the mean±SD of three independent experiments of real-time qPCR. GAPDH is a negative control. ***P<0.001. (D) Knockdown of PTBP1 in KYSE 30 cells. Three siRNAs were used to knock down the PTBP1. (E) Dual-luciferase reporter assay after knockdown of PTBP1. The activity of IRES was measured after the knockdown of PTBP1. The ratio of FLuc and RLuc indicates the activity of the IRES. Data are presented as the mean±SD of five independent experiments. *P<0.05.