Journal: bioRxiv

Article Title: The nuclear sulfenome of Arabidopsis : spotlight on histone acetyltransferase GCN5 regulation through functional thiols

doi: 10.1101/2024.04.24.590918

Figure Lengend Snippet: Predicted structure of Arabidopsis GCN5 and experimental evidence for its oxidative post-translational modification. (A) Schematic diagram of the GCN5 domains and positions of the individual cysteine residues. (B) Multi-domain homology model of GCN5 according to MODELLER. (C) AlphaFold2 prediction of the GCN5 structure. Coloured based on pLDDT score. (D) Sulfenylation of recombinant His/MBP-tagged GCN5 protein upon H 2 O 2 treatment. Recombinant GCN5 protein was incubated with increasing H 2 O 2 concentrations, loaded on a gel and visualised with anti-cysteine sulfenic acid antibody. Ponceau staining shows the loading control. His/MBP-tagged GCN5 migrated as a single band at ∼106 kDa. (E) Histone acetyltransferase activity of recombinant HisMBP-GCN5 in the presence of H 2 O 2.

Article Snippet: A colorimetric histone acetyltransferase activity assay kit (ab65352, Abcam; Cambridge, MA, USA) was used to measure in vitro HAT activity according to the manufacturer’s instructions.

Techniques: Modification, Recombinant, Incubation, Staining, Activity Assay

Journal: iScience

Article Title: Small-molecule TIP60 inhibitors enhance regulatory T cell induction through TIP60-P300 acetylation crosstalk

doi: 10.1016/j.isci.2023.108491

Figure Lengend Snippet: TIP60 inhibitors increase HAT and KAT activity at low-mid concentrations (A) Description of the peptide-based assay to determine TIP60 histone acetyltransferase (HAT) and Foxp3 lysine acetyltransferase (KAT) activities. (B and C) Dose-response curve for the effect of TIP60 inhibitors NU9056, MG149, and TH1834 on TIP60 B HAT and C KAT activities. (D) Baseline TIP60 and P300 KAT activity on Foxp3 and (E) differential effect of TIP60 inhibitors and P300 inhibitor C646 (20 μM) on P300 and TIP60 KAT activity (mean ± SEM, n = 3–5 from at least two independent experiments, B, C, and E one-way ANOVA followed by Tukey post-hoc test, all comparisons with normalized baseline; D mean ± SEM, t-test; ∗p < 0.05, #p < 0.01).

Article Snippet: Histone Acetyltransferase (HAT) Activity Assay Kit , SigmaAldrich , Cat#EPI001-1KT.

Techniques: Activity Assay

Journal: iScience

Article Title: Small-molecule TIP60 inhibitors enhance regulatory T cell induction through TIP60-P300 acetylation crosstalk

doi: 10.1016/j.isci.2023.108491

Figure Lengend Snippet: TIP60 inhibitors modulate P300 acetyltransferase activity on Foxp3 (A) FRET-based strategy to measure TIP60/P300 heteromer formation and representative histograms indicating FRET signal in CD4 + Foxp3 + cells from Treg cell induction cultures treated with or without TIP60 inhibitors (NU9056 20 μM, MG149 and TH1834 25 μM) and (B) FRET-Efficiency summary results. (C) Representative scatterplots and summary results indicating the percentage of Foxp3 + cells at the end of Treg induction cultures in the presence of MG149 (20 μM), C646 (1μM), or MG149 + C646. (D–F) Summary results of TIP60 acetylation, TIP60/P300 heteromer formation, P300 acetylation, and Foxp3 acetylation measured by FRET (FRET Efficiency) in identical cultures treated with MG149 (D), MG149 + C646 (E) or C646 alone (F). (Bar graphs depict mean ± SEM, n = 4 from three independent experiments. ANOVA followed by post-hoc Tukey HSD test. (D–F) The ANOVA was performed for the same FRET pair across Vehicle, MG149, C646, and MG149 + C646 groups and all comparisons referred to vehicle; ∗p < 0.05, #p < 0.01).

Article Snippet: Histone Acetyltransferase (HAT) Activity Assay Kit , SigmaAldrich , Cat#EPI001-1KT.

Techniques: Activity Assay

Journal: iScience

Article Title: Small-molecule TIP60 inhibitors enhance regulatory T cell induction through TIP60-P300 acetylation crosstalk

doi: 10.1016/j.isci.2023.108491

Figure Lengend Snippet:

Article Snippet: Histone Acetyltransferase (HAT) Activity Assay Kit , SigmaAldrich , Cat#EPI001-1KT.

Techniques: Recombinant, HAT Activity Assay, Lysis, Bicinchoninic Acid Protein Assay, Membrane, Western Blot, SYBR Green Assay, Cell Isolation, Selection, In Situ, Staining, Software