Journal: Epigenetics
Article Title: Aurora kinase a phosphorylates and stabilizes UHRF1 to maintain DNA methylation and prostate cancer cell survival
doi: 10.1080/15592294.2026.2719245
Figure Lengend Snippet: AURKA physically interacts with the UHRF1 protein and induces its phosphorylation. (A) Immunoprecipitation was conducted using PC3 cells expressing AURKA-Flag, and the experimental samples were analyzed via mass spectrometry. (B-C) Cell lysates underwent immunoprecipitation with either anti-AURKA or anti-UHRF1 antibody. Subsequently, the immunoprecipitated protein was subjected to immunoblotting with the specified antibodies. (D) Recombinant GST and GST-UHRF1 proteins were purified. The protein interaction between UHRF1 and AURKA was then investigated through a GST-pull down assay. (E) Bacterial-expressed GST-UHRF1 was subjected to an in vitro kinase assay, and the phosphorylation level of UHRF1 was evaluated using an anti-phosphoserine antibody. The loading controls are presented in the bottom panel (stained by Coomassie blue). (F) Schematic representation of the putative AURKA-mediated phosphorylation sites in the UHRF1 protein. (G) Bacterial-expressed GST-UHRF1 wild-type or mutants were subjected to an in vitro kinase assay, and the phosphorylation level of UHRF1 was evaluated using an anti-phosphoserine antibody. The loading controls are presented in the bottom panel (stained by Coomassie blue). (H) Cells were transfected with plasmids expressing AURKA-Flag and UHRF1-His wild type and mutants. UHRF1 was immunoprecipitated with anti-His antibody, and the total phosphorylation level was measured with an anti-phosphoserine antibody.
Article Snippet: The antibodies and chemicals employed in this study included anti-Flag (Sigma, F1804; Cell Signal Technology, CST, 14,793), anti-His (Genscript, A00186), anti-phosphoserine (Santa Cruz Biotechnology, Santa, sc-81516), anti-AURKA (CST, 14,475; Proteintech, 66,757–1-Ig), anti-UHRF1 (Proteintech, 21,402–1-AP; Santa, sc-373750), anti-HA (CST, 3724), anti-5-Methylcytosine (Active motif, 39,649; CST, 28,692), and anti-β-actin (Abclonal, AC004).
Techniques: Phospho-proteomics, Immunoprecipitation, Expressing, Mass Spectrometry, Western Blot, Recombinant, Purification, GST Pulldown Assay, In Vitro, Kinase Assay, Staining, Transfection
