Fig. 1: MED1 is acetylated under the control of p300/CBP.
From: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment
a, Immunoblotting showing enhanced lysine acetylation (referred to as AceK) of FLAG–MED1 in HEK293T cells following overexpression of WT but not D1399Y mutant p300 (human and mouse, with 98% sequence identity to human CBP). IB, immunonlot. b, Immunoblotting showing enhanced acetylation of FLAG–MED1 in HEK293T cells following overexpression of p300 and its suppression by the p300 inhibitor A485 but not C646 or SGC-CBP30. Cells were treated with these inhibitors at the concentrations indicated for 24 h. c, Immunoblotting showing enhanced acetylation of FLAG–MED1 following overexpression of p300 or CBP but not GCN5, PCAF, TIP60 and MOF in HEK293T cells. d, Immunoblotting showing enhanced acetylation of endogenous MED1 in HEK293T cells following overexpression of p300 or CBP. e, Immunoblotting showing enhanced acetylation of endogenous MED1 from HEK293T cells with overexpression of p300 at variable concentrations. f, Immunoblotting showing enhanced acetylation of MED1 (within the purified Mediator complex) by p300 and acetyl-CoA. Effects of variable p300 concentrations (top) and variable incubation times (bottom) were tested. Autoacetylation of p300 was also observed. g, Signal of digested peptides with acetylation plotted as a function of amino acid sequence. Signals from control samples and from p300 overexpression samples with or without A485 were compared and the two acetylation clusters are indicated. h, Analysis of MED1 protein sequence by MobiDB showing a disordered region (amino acids 947–1573). i, Immunoblotting showing enhanced acetylation of WT but not 6KR FLAG–MED1 in HEK293T cells following overexpression of p300. Effects of variable p300 concentrations were tested. For d,e, IgG was used as an IP control. For a–e,i, densitometry analysis was performed on immunoblotting images and the normalized protein levels (relative to the IP target protein, either FLAG-tagged or endogenous MED1) are shown. For a–f,i, immunoblots are representative of three independent experiments.
