Figure 3

DN-DBC1 lacks the first 124–138 residues and maintains the LZ domain while not binding to SIRT1. (A) DBC1 (upper panel) and DN-DBC1 (lower panel) were purified by immunoprecipitation from mice livers and digested with trypsin previous to the analysis by mass spectrometry (MS). Upper panel, partial Mass Spectrometry (MS) spectrum of Full-length (FL) protein. Lower panel, partial MS spectrum of DN protein. Arrow in inset show the peak corresponding to peptides that were not detected in DN-DBC1 (B) Scheme of DBC1 sequence showing the different domains and the proposed site of cleavage. (C) Alignment of the first 188 amino acids of mouse and human DBC1. The region found to be lost in DN-DBC1 is highlighted in yellow, showing 100% aminoacidic residues conservation among species. (D) Flag-tagged FL and DN-DBC1 were overexpressed in 293 T cells and their binding to endogenous SIRT1 was evaluated by co-immunoprecipitation. Non-transfected cells were used as controls. A representative experiment out of three independent experiments is shown. (E) SIRT1 activity in 293 T cells transfected with empty vector (−) Flag-tagged FL, or Flag-tagged DN-DBC1. Activity was expressed as a percent of control (−) cells. *means p < 0.05 compared to control (−). One-way ANOVA. (F) Flag-tagged FL, DN-DBC1 and SIRT1 were overexpressed in 293 T. SIRT1 activity regulation was evaluated by H3 acetylation in Lysine 9 (AcH3K9). A representative western blot is shown. (G) SIRT1 and DBC1 interaction assayed by pull-down experiments. Flag-tagged recombinant SIRT1 was purified from overexpression in 293 T cells. 293 T cell lysates were incubated with liver lysates mainly expressing DBC1 (FL) or DN-DBC1 (DN). Pull-down assays were performed by incubating with Flag antibody (left panels) or DBC1 (C-terminus) antibody (right panels). Input is shown in the center. A representative experiment out of three independent ones is shown. Black and red arrows point to DBC1 and DN-DBC1 respectively.