Journal: Molecular and Cellular Biology

Article Title: Corepressive Action of CBP on Androgen Receptor Transactivation in Pericentric Heterochromatin in a Drosophila Experimental Model System

doi: 10.1128/mcb.02123-07

Figure Lengend Snippet: FIG. 6. dSir2 is acetylated by dCBP in vivo and in vitro at its conserved HDAC catalytic core region. (A and B) dCBP human homologue (p300) or p300/CBP HAT acetylates Sir2 in vitro. The p300, GST-P300/CBP HAT, and GST-PCAF HAT proteins were subjected to in vitro acetylation assays with in vitro-synthesized substrate proteins, including mSir2, human HP1, and SUV39H1. Reaction products were analyzed by autora- diography (14C). The autoacetylated p300 or p300/CBP HAT is indicated with a star. Fractions of 35S-labeled in vitro-synthesized substrate proteins (2.5%) were used as the input (panel A, lane 1; panel B, lanes 1 and 5). (C) (Upper panel) Schematic representation of Sir2 orthologues in Drosophila flies (dSir2); the conserved lysine sites at its catalytic core regions (SIRT1 domain) are indicated with stars. H331 is a well-defined catalytic site of dSir2 (open arrowhead). (Lower panel) Sequence alignment of the conserved core region with the SIRT domains from several species as indicated. Conserved residues with 100% identity are indicated with red characters. Below the alignment, conserved lysine (K) residues

Article Snippet: The cell lysates were harvested and immunoprecipitated with anti-acetylated lysine antibody (Ac-K103 [catalog no. 9681[; Cell Signaling Technology).

Techniques: In Vivo, In Vitro, Synthesized, Labeling, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Corepressive Action of CBP on Androgen Receptor Transactivation in Pericentric Heterochromatin in a Drosophila Experimental Model System

doi: 10.1128/mcb.02123-07

Figure Lengend Snippet: FIG. 8. The acetylation of mSir2 by CBP is mediated by the association between mSir2 and CBP. (A) Schematic representation of mSir2 and its truncation mutations. (B) Anti-FLAG IP was performed on extracts from 293T cells expressing FLAG-HA-mSir2 or its truncation mutations. Immunoprecipates (IP) prepared using anti-FLAG and whole-cell extracts (Input) were analyzed by immunoblotting using the indicated anti- bodies. Binding of CBP to mSir2 fragments is lost when both the N and C termini of mSir2 are truncated (SIRT) (lane 6), suggesting that both the N and C termini of mSir2 are required for the association between mSir2 and CBP. (C) 293T cells were cotransfected with FLAG-HA-mSir2 wt or a series of truncation mutation expression plasmids together with CBP expression vector as indicated. The whole-cell lysates were immunoprecipitated with anti-acetylated lysine antibody (-Ac-K). Precipitated proteins and fractions (5%) of the input cell lysates were analyzed by immunoblotting (IB) with anti-FLAG or anti-HA antibodies.

Article Snippet: The cell lysates were harvested and immunoprecipitated with anti-acetylated lysine antibody (Ac-K103 [catalog no. 9681[; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Binding Assay, Mutagenesis, Plasmid Preparation, Immunoprecipitation

Journal: iScience

Article Title: Mitochondrial hyper-acetylation induced by an engineered acetyltransferase promotes cellular senescence

doi: 10.1016/j.isci.2025.113233

Figure Lengend Snippet: Global mitochondrial acetylation controlled by eMAT and SIRT3 (A) A schematic for quantitative LC-MS/MS. Mitochondria fractions from parental, eMAT treated with 100 ng/mL Dox, eMAT+SIRT3 treated with 100 ng/mL Dox were isolated and treated with trypsin. The tryptic peptides were immunoprecipitated with anti-AcK antibodies, and captured with Protein-A/G agarose beads. The beads-bound peptides were eluted and analyzed with LC-MS/MS with a label-free quantitative method (Proteome Discoverer Ver.3.1). (B) Venn diagram of identified AcK-containing peptides. (C) Violin plot of the AcK peptides. Friedman Test: p = 4.295 × 10 −5 . (D) Comparison of acetylation between eMAT and control sample. Fold change of acetylated peptides (total 1240 peptides) was calculated as log2([peptide abundance of eMAT]/[peptide abundance of control]). (E) Ven diagram of the eMAT targets. Among 725 identified substrates with log2FC > 1, 74.3% were known proteins. (F) Consensus motif analysis of acetylation sites with WebLogo (Ver.3). GO analysis of eMAT targets with DAVID (Ver.2021): (G) Cellular compartment, (H) Biological process. (I) Comparison of acetylation between eMAT+SIRT3 and eMAT. Fold changes were calculated as log2([peptide abundance of eMAT+SIRT3]/[peptide abundance of eMAT]). (J) Ven diagram of the eMAT and SIRT3 targets. (K) Consensus motif analysis of deacetylation sites by SIRT3. GO analysis of eMAT and SIRT3 targets: (L) Cellular compartment, (M) Biological process.

Article Snippet: Acetylated Lysine (Ac-K2-100) MultiMab Rabbit mAb mix , Cell Signaling Technology , Cat#9814; RRID:AB_10544700.

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Immunoprecipitation, Comparison, Control

Journal: iScience

Article Title: Mitochondrial hyper-acetylation induced by an engineered acetyltransferase promotes cellular senescence

doi: 10.1016/j.isci.2025.113233

Figure Lengend Snippet: eMAT acetylates multiple mitochondrial metabolic enzymes and SOD2 (A) Validated metabolic enzymes acetylated by eMAT: Orange; eMAT-dependent acetylation, magenta; eMAT-dependent acetylation and SIRT3-dependent deacetylation. FLAG-tagged proteins were expressed in Dox-inducible eMAT cells (B) or eMAT+Vec and eMAT+SIRT3 cells (C), treated with 10 ng/mL Dox for 24 h. Immunoprecipitated proteins were blotted with anti-AcK antibodies or anti-FLAG antibodies. Expression of eMAT (anti-V5) and SIRT3 (anti-HA) was confirmed with immunoblot of total cell lysates. LC-MS/MS analysis of FLAG-tagged (D) ACO2, (E) DLST, (F) SDHA, (G) SUCLG1 in control (eMAT-V5+vec cells without Dox treatment), eMAT (eMAT-V5+vec cells treated with 10 ng/mL Dox for 24 h), eMAT+SIRT3 (eMAT-V5+SIRT3 treated with 10 ng/mL Dox for 24 h) cells. Normalized acetylation (%) was calculated based on the intensity of the acetylated peptide, normalized by the sum of the intensities of the corresponding unacetylated and acetylated peptides. Blue; common acetylation site whose acetylation was detected both in the control and eMAT. (H) Schematic of eMAT mediated acetylation of metabolic enzymes and SOD2. (I) Representative immunoblot of eMAT-dependent acetylation of SOD2. (J) Quantitation of SOD2 acetylation. n = 5; mean ± SEM. Tukey’s HSD test: p † < 0.1, p ∗ < 0.05. (K) LC-MS/MS analysis of FLAG-tagged SOD2 in control, eMAT, eMAT+SIRT3 cells. (L) Venn diagram showing the number of acetylation sites identified in control and eMAT conditions across the five substrates.

Article Snippet: Acetylated Lysine (Ac-K2-100) MultiMab Rabbit mAb mix , Cell Signaling Technology , Cat#9814; RRID:AB_10544700.

Techniques: Immunoprecipitation, Expressing, Western Blot, Liquid Chromatography with Mass Spectroscopy, Control, Quantitation Assay