Journal: The Journal of Biological Chemistry
Article Title: Identification of Akt Interaction Protein PHF20/TZP That Transcriptionally Regulates p53
Figure Lengend Snippet: PHF20 binds to DNA and transcriptionally up-regulates p53. A, association of PHF20 with chromatin. Cells were resuspended in buffer A with Nonidet P-40 (0.1%). Nuclei were collected by low speed centrifugation. Insoluble chromatin pellet (P3) was collected by centrifugation and resuspended in SDS-loading buffer. Release of chromatin-bound PHF20 proteins was detected following treatment of the cell nuclei with micrococcal nuclease ( MNase ). B, electrophoretic mobility shift assay was performed as described under “Experimental Procedures.” C, PHF20 possesses transcription factor activity. Three repeats of PHF20-binding consensus binding sequence were cloned to pGL3-basic vector ( 3 × PBCS-Luc ), which was co-transfected into MCF7 cells with indicated plasmids. Following incubation for 36 h, luciferase activity was measured and normalized to β-galactosidase. Results are the mean ± S.E. of three independent experiments performed in triplicate. D and E, PHF20 induces p53. MCF7/Tet-Off inducible PHF20 cells were cultured in the media without doxycycline ( Dox ) for the indicated time and then were immunoblotted with indicated antibodies ( D ) and were subjected to Northern blot analysis probed with p53 and β-actin ( E ). F, knockdown of PHF20 reduces p53 expression. MCF7 and HCT116 were transfected with PHF20 siRNA or control siRNA and subjected to immunoblotting ( left ) and Northern blot ( right ) analysis. G, PHF20 induces p53 promoter activity. Luciferase report assay was performed as described in C in MCF7 cells that were transfected with indicated plasmids. H, ChIP assay shows that PHF20 directly binds to p53 promoter in vivo . ChIP assay was performed with PHF20 antibody in HCT116 cells, and PCR was carried out using the primers spanning two PHF20-binding sites in the p53 promoter. IP , immunoprecipitation.
Article Snippet: To release chromatin-bound proteins by nuclease treatment, cell nuclei were resuspended in prewarmed buffer A plus 1 m m CaCl2 and 0.3 units of micrococcal nuclease (Sigma).
Techniques: Centrifugation, Electrophoretic Mobility Shift Assay, Activity Assay, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Cell Culture, Northern Blot, Expressing, Chromatin Immunoprecipitation, In Vivo, Polymerase Chain Reaction, Immunoprecipitation