Myc-tagged IPMK (mycIPMK) and treated overnight with 20 M etoposide (eto) were lysed and subjected to immunoprecipitation (IP) with antimyc agarose. IPMK and p53 were resolved in the immunoprecipitated samples by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and identified by Western blotting (WB). Inputs for p53, mycIPMK, and actin are shown in the bottom blots. (B) Wild-type major MEFs treated with 20 M etoposide have been lysed and subjected to immunoprecipitation with normal immunoglobulin G (IgG) or antibody precise for IPMK. SDS-PAGE and Western blotting were performed as described in (A). (C) Lysates from IPMK-deficient (/) and floxed wild-type (fl/fl) MEFs were employed as described in (B) to assess endogenous IPMK-p53 binding. (D) Immunoprecipitation of purified mycIPMK and bacterially purified p53, as assessed by immunoprecipitation with anti-myc agarose and subsequent protein resolution and identification by SDS-PAGE and Western blotting. Blots in all panels are representative of at least three experiments.Eptifibatide Sci Signal. Author manuscript; out there in PMC 2014 July 23.Xu et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. two.IPMK augments the transcriptional activity of p53. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis (left) and quantification (correct) of PUMA, Bax, and p21 mRNAs in U2OS cells transfected with plasmids encoding myc or mycIPMK and treated overnight with ten M etoposide.Miglustat Data are signifies SEM from 3 experiments.PMID:23983589 ***P 0.001, Student’s t test. (B) Western blotting evaluation of PUMA, Bax, and p21 proteins in HCT116 cells transfected and treated as in (A). Information are implies SEM from three experiments. *P 0.05, **P 0.01, Student’s t test. (C) Western blotting evaluation of PUMA, Bax, and p21 proteins in U2OS cells transfected and treated as in (A). Information are means SEM from three experiments. *P 0.05, **P 0.01, Student’s t test. (D) qRT-PCR analysis (left) and quantification (appropriate) of PUMA, Bax, and p21 mRNAs in fl/fl or /Sci Signal. Author manuscript; out there in PMC 2014 July 23.Xu et al.PageMEFs treated overnight with 20 M etoposide. Data are indicates SEM from three experiments. ***P 0.001, n = 3, imply SEM, Student’s t test.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; readily available in PMC 2014 July 23.Xu et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal. Author manuscript; available in PMC 2014 July 23.Fig. three.IPMK enhances p53 localization to target promoters and augments expression of p53 downstream targets. (A) Western blotting evaluation of p53 and its targets PUMA, Bax, and p21 in fl/fl or / MEFs treated overnight with 20 M etoposide. **P 0.01, n = three, mean SEM, Student’s t test. (B) Amounts of p21, PUMA, and Bax mRNAs in wild-type (+/+) and p53-null (-/-) HCT116 cells. ***P 0.001, n = 3, imply SEM, Student’s t test. (C) Detection of IPMK at the promoters or exon regions of PUMA, Bax, and p21 by ChIP analysis of lysates from etoposide-treated wild-type MEFs. Data are implies SEM from 3 experiments. ***P 0.001, Student’s t test. (D) ChIP analysis of p53 binding for the promoter regions of PUMA, Bax, and p21 in etoposide-treated fl/fl or / MEFs. Data are signifies SEM from three experiments. ***P 0.001, Student’s t test.Xu et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 4.IPMK stimulates p53 acetylation and.