Research Paper Volume 13, Issue 8 pp 11822—11832

ITPKA induces cell senescence, inhibits ovarian cancer tumorigenesis and can be downregulated by miR-203

ITPKA interacted with MDM2. (A) GST pull-down assays were performed to examine the interaction between the fusion proteins GST-MDM2 and ITPKA. OVCAR3 cell lysates were used. (B) Immunoprecipitation assay was performed to examine the interaction between flag-tagged MDM2 and myc-tagged ITPKA. Flag-MDM2 and myc-ITPKA were transfected into OVCAR3 cells. Forty-eight hours later, the cells were harvested, and immunoprecipitation assays were performed using an anti-Flag antibody. (C) Immunoprecipitation assay was performed to examine the interaction between endogenous MDM2 and ITPKA. Protein from OVCAR3 cells was harvested, and immunoprecipitation assays were performed using an anti-MDM2 antibody. (D–E) Stability of P53 was examined after the cells were treated with CHX at the indicated time points. (F) mRNA level of P21 was examined using q-PCR. (G) Immunoprecipitation was performed to examine the interaction between P53 and MDM2 in OVCAR3 cells. (H) Ubiquitination of P53 was examined in OVCAR3 cells. (I) P53 knockdown abolished the function of ITPKA in cell senescence. **P ##P

Figure 4. ITPKA interacted with MDM2. (A) GST pull-down assays were performed to examine the interaction between the fusion proteins GST-MDM2 and ITPKA. OVCAR3 cell lysates were used. (B) Immunoprecipitation assay was performed to examine the interaction between flag-tagged MDM2 and myc-tagged ITPKA. Flag-MDM2 and myc-ITPKA were transfected into OVCAR3 cells. Forty-eight hours later, the cells were harvested, and immunoprecipitation assays were performed using an anti-Flag antibody. (C) Immunoprecipitation assay was performed to examine the interaction between endogenous MDM2 and ITPKA. Protein from OVCAR3 cells was harvested, and immunoprecipitation assays were performed using an anti-MDM2 antibody. (DE) Stability of P53 was examined after the cells were treated with CHX at the indicated time points. (F) mRNA level of P21 was examined using q-PCR. (G) Immunoprecipitation was performed to examine the interaction between P53 and MDM2 in OVCAR3 cells. (H) Ubiquitination of P53 was examined in OVCAR3 cells. (I) P53 knockdown abolished the function of ITPKA in cell senescence. **P < 0.01; ##P < 0.01.