Histone H3 acetylation by recruiting p300. (A) Immunoprecipitation and Western blotting

Histone H3 acetylation by recruiting p300. (A) Immunoprecipitation and Western blotting had been applied to detect binding between p300 and p53 in lysates from HCT116 cells transfected with plasmids encoding myc or mycIPMK and treated with 10 M etoposide overnight. Information are implies SEM from three experiments. **P 0.01, Student’s t test. (B) Immunoprecipitation for acetylated lysine (Ac-Lys) and Western blotting for acetylated p53 at Lys373 (Ac-Lys373) and Lys382 (Ac-Lys382) in lysates from etoposide-treated HCT116 cells transfected with plasmids encoding myc or mycIPMK. Data are means SEM from 3 experiments. **P 0.01, *P 0.05, Student’s t test. (C) Immunoprecipitation and Western blotting had been utilised to detect acetylated p53 and p300-p53 binding in lysates from etoposide-treated HCT116 cells transfected with either control shRNA or IPMK-specific shRNA. Information are indicates SEM from four experiments.Umeclidinium bromide ***P 0.001, Student’s t test. (D) Western blotting and quantification of your acetylation of purified p53 (Ac-Lys) when exposed to combinations of purified IPMK and p300 within the presence of acetyl-CoA (coenzyme A). Data are indicates SEM from three experiments. ***P 0.001, Student’s t test. (E) ChIP evaluation of acetylation (Ac-H3K9 compared with H3) and p300 binding (p300 compared with IgG) at the promoter area of p21 in etoposide-treated fl/fl and / MEFs. Data are means SEM from 3 experiments. ***P 0.001, Student’s t test.Sci Signal. Author manuscript; readily available in PMC 2014 July 23.Xu et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 5.IPMK mediates cell death. (A) Flow cytometry evaluation with annexin V ITC (fluorescein isothiocyanate) and propidium iodide (PI) of reside (reduced left quadrant), dead (upper appropriate), and apoptosing (reduce correct) U2OS cells transfected with plasmids encoding myc or mycIPMK and treated with 20 M etoposide. (B and C) DNA fragmentation (B) and Western blot evaluation (C) of PARP and caspase-3 cleavage in transfected, etoposide-treated U2OS cells. Information are signifies SEM from three experiments. **P 0.01, *P 0.05, Student’s t test. (D to F) Analysis of cell viability (D), DNA fragmentation (E), and PARP and caspase-3 cleavage (F) in fl/fl and / MEFs treated with 20 M etoposide. Data are indicates SEM from three experiments. ***P 0.001, **P 0.01, Student’s t test. (G) Western blot analysis of PARP and caspase-3 cleavage in HCT116 cells transfected with handle or hIPMK shRNA and treated with 120 M sulindac. Data are means SEM from 3 experiments (ns, no significance; Student’s t test). (H) Cell proliferation was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay in wildtype (+/+) and p53-null (-/-) HCT116 cells transfected with handle or hIPMK-specific shRNA and treated with 10 M etoposide.Chymotrypsin Information are implies SEM from three experiments.PMID:24189672 **P 0.01, Student’s t test.Sci Signal. Author manuscript; available in PMC 2014 July 23.Xu et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 6.Overexpression of a fragment of IPMK inhibits endogenous IPMK function. (A) Immunoprecipitation and Western blotting analysis have been utilised to detect binding between p53 and fragments of IPMK in p53-null (-/-) HCT116 cells cotransfected with plasmids encoding GST, GST-tagged full-length (FL) IPMK, or GST-tagged exon-encoded IPMK fragments, he-magglutinin (HA) agged p53, or myc-tagged p53. Left, whole-cell lysates (WCLs); proper, immunoprecipitate frac.