Extended Data Fig. 3: MED1 acetylation is regulated by p300/CBP and identification of acetylation sites in the MED1 IDR.
From: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment
a, b, Detection of acetylation of FLAG-tagged Mediator subunits transiently expressed in HEK293T cells treated with Trichostatin A (TSA, 0.5 μM for 24 h) and nicotinamide (NAM, 5 mM for 3/6 h), referred to as T + N3 or T + N6 in the panel. Immunoblotting images (a) and densitometry quantification with AceK immunoblot signals normalized by the FLAG signals (b) are shown. c, Silver staining (left) and immunoblotting (right) confirming the yield and integrity of Mediator (presence of MED1 and MED17) purified from HeLa-S cells and the absence of p300 and SIRT1. d, Workflow for mass spectrometry identification of acetylation sites from immunoprecipitated endogenous MED1 from HEK293T cells with overexpression of p300 with or without A485. e, Immunoblotting confirming the overexpression of p300 and acetylation of MED1 in IP-Mass Spectrometry samples. Densitometry analysis was performed on immunoblotting images, and the normalized acetyl-lysine levels (relative to IP target protein, MED1) are shown. f, Analysis of MED1 protein sequence by VL-XT algorithm showing a disordered region (963-1320 aa) with high PONDR score. g, Multiple sequence alignments indicating that the 6 acetylation sites (arrows) in the MED1 IDR are conserved among species. h, Immunostaining with anti-FLAG antibodies combined with DAPI staining for MCF7 cells with ectopic expression of FLAG-tagged WT or 6KR MED1. Scale bar is 20 μm. For panels a (MED1 test), c, e, h, the results are representative of three independent experiments.
