Figure 4.

circ-Amotl1 interacted with c-myc protein. (a) MDA-MB-231 cell lysate was incubated with antibodies as indicated. Anti-c-myc, EGF, c-myb, NF1, and AKT antibodies pulled down circ-Amotl1 but not linear Amotl1 mRNA. **P<0.01. Error bars, S.D. (n=4). (b) Lysates prepared from vector- or circ-Amotl1-transfected MDA-MB-231 cells were incubated with antibodies as above, followed by real-time PCR. Anti-c-myc, EGF, c-myb, NF1, and AKT antibodies pulled down significantly higher levels of circ-Amotl1 in the circ-Amotl1-transfected cells relative to the control. *P<0.05, **P<0.01. Error bars, S.D. (n=4). (c) Equal amounts of proteins were detected in the vector- and circ-Amotl1-transfected cells. (d) The lysates were also incubated with antibodies as indicated, followed by real-time PCR. Anti-c-myc antibody pulled down circ-Amotl1 but not circ-Foxo3. (n=4). (e) Cell lysates prepared from MDA-MB-231 cells were subjected to immunoprecipitation, followed by real-time PCR. Antibodies against c-myc pulled down circ-Amotl1 but not the other circRNAs. (n=4). (f) MDA-MB-231 cell lysate was incubated with different antibodies followed by real-time PCR. Anti-c-myc antibody pulled down circ-Amotl1 but not linear Amotl1 mRNA. (n=4). (g) Lysates prepared from vector- or circ-Amotl1-transfected (including circ-Amotl1-m and circ-Amotl1-c) cells were incubated with antibodies as above, followed by real-time PCR. Anti-c-myc antibody pulled down circ-Amotl1 but not Amotl1 mRNA. (n=4). (h) The mixes were incubated with streptavidin beads to pull down circ-Amorl1. Real-time PCR showed that the probe of circ-Amotl1 pulled down significantly higher levels of circ-Amotl1 in cells transfected with circ-Amotl1 than in the control cells. (n=4). (i) Protein lysates prepared from panel (h) were subject to western blotting. The probe complementary to circ-Amotl1 pulled down more c-myc than the control