Fig. 5

Identification of the binding sites of circYap with Yap mRNA. a The binding sites of circYap with Yap mRNA were identified by using the RISearch software. The 2′-O-methyl blocking oligos with complementary sequences were designed. Blocking oligos for the two binding sites (Block-1 and Block-2) or the siRNA for Yap mRNA (siYap-1 and siYap-2) were transfected to MDA-MB231 cells which were then collected 48 h after transfection. The cell lysates from negative control (NC), blocking oligo, or siYap groups were incubated with biotinylated circYap probe or Yap mRNA probe and streptavidin beads. The RNAs were eluted by Trizol reagent from the beads for detecting Yap mRNA and circYap pulled down by probes. The proteins were eluted by RNase-free water containing 0.1% SDS to examining the PABP and eIF4G protein pulled down by probes. b The circYap (left) and Yap mRNA (right) levels after pull down by the probes were examined by real-time PCR. n = 4. **p < 0.01 compared to negative control (NC). c PABP or eIF4G that were pulled down by circYap probes (left) or Yap probes (right) were examined by western blotting. d Lysates prepared from cells transfected with negative control (NC), blocking oligo, or siYap were incubated with PABP or eIF4G antibodies and protein A magnetic beads to examine the precipitated circYap (left) and Yap mRNA (right) by real-time PCR. n = 4. **p < 0.01 compared to negative control. e Yap expression in MDA-MB231 cells transfected with NC and blocking oligos, or transfected with vector or Yap plasmid were examined by western blotting