Fig. 3

Binding of circYap with PABP and eIF4G protein. a, b The cell lysate from MDA-MB231 wide-type cells, the cells stably transfected with vector, circYap or its linear precursor (linear Yap), or angiomotin like-1 circRNA (circAmotl1) were incubated with antibody against rabbit or mouse IgG, PABP or eIF4G, and protein A magnetic beads to precipitate RNAs followed by real-time PCR with primers specific for circYap (a) or linear Yap mRNA (b). n = 6. **p < 0.01 compared to corresponding vector control. c The expression of PABP and eIF4G were examined in the lysates from MDA-MB231 cells stably transfected with or without vector, circYap, linYap, circAmotl1, or Ccnb1 circRNA (circCcnb1). d Lysates from MDA-MB231 cells transfected with si-ctrl or si-circYap were incubated with antibodies against PABP antibodies (left) or eIF4G (right) followed by real-time PCR. n = 6. **p < 0.01 compared to siRNA control. e MDA-MB231 cell lysates were incubated with biotinylated circYap probe or scramble oligo, and streptavidin beads. The PABP and eIF4G proteins that were pulled down by circYap probe were analyzed by western blotting. n = 3. f Lysates from MDA-MB231 cells transfected with vector or circYap were incubated with circYap probe or Yap mRNA probe. The pulled down proteins were probed by antibodies against PABP (left) and eIF4G (right). n = 3. g Left, protein extract from MDA-MB231 cells stably transfected with or without vector and circYap were used for immunoprecipitation (IP) followed by immunoblotting (IB) analysis. Right, after immunoprecipitation (IP) with either PABP or eIF4G antibody, 1/3 of the protein-bound magnetic beads from each group were treated with RNase R to digest linear RNAs and another 1/3 were treated with RNase A to digest both linear and circular RNAs. Then, the beads were washed and the precipitated proteins were eluted with Laemmli buffer followed by western immunoblotting (IB). n = 3