Journal: Cell Cycle

Article Title: Epidermal Growth Factor Receptor activation promotes ADA3 acetylation through the AKT-p300 pathway

doi: 10.1080/15384101.2017.1339846

Figure Lengend Snippet: The phospho defective mutant p300 S1834A has reduced ability to acetylate ADA3. (A) HEK-293T cells were transfected with HA-tagged wild-type or S1834A/E mutants of p300. 48 h after transfection, whole cell extracts were subjected to immunoprecipitation by agarose conjugated anti-HA beads. Immunoprecipitates were then eluted with HA peptide and visualized on SDS gels by CBB staining. (B & C) In vitro KAT assay using 20 ng HA-tagged wild type or S1834A/E p300 mutants as enzymes obtained from A, and 1 μg of GST-ADA3 (B) or recombinant histone H3 (C) as substrates, followed by immunoblotting with anti-acetyl lysine antibody. The numbers underneath anti-Ac lysine blot in (B) represents the band intensity of GST-ADA3 acetylation normalized over total GST-ADA3 in ponceau stain as computed from ImageJ software. (D) HEK-293T cells were co-transfected with FLAG-tagged ADA3 and HA-tagged wild- type p300 or S1834A mutant. 48 h after transfection whole cell extracts were immunoprecipitated with M2 agarose and then immunoprecipitates were eluted by 3x FLAG peptide, followed by immunoblotting with indicated antibodies.

Article Snippet: Anti-acetyl lysine (9681), anti-acetyl lysine-HRP (6952) and anti-HA (2999) antibodies were from Cell Signaling Technologies.

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Staining, In Vitro, Recombinant, Western Blot, Software