Fig. 7: MED1-IDR droplets compartmentalize and concentrate proteins necessary for transcription.

(A) MED1-IDR droplets incorporate BRD4-IDR protein in vitro. The indicated mEGFP or mCherry fusion proteins were mixed at 10μM each in Buffer D containing 10% Ficoll-400 and 125mM NaCl. Indicated fluorescence channels are presented for each mixture. Illustrations summarizing results shown on left. (B) MED1-IDR forms droplets in an in vitro transcription reaction containing HeLa cell nuclear extract, while the MED1-IDR S-to-A mutant does not. Representative images of indicated mEGFP-fusion protein when added to an in vitro transcription reaction containing HeLa cell nuclear extract at a final concentration of 3mg/ml (see Materials and Methods for complete list of components). (C) MED1-IDR droplets compartmentalize transcriptional machinery from a nuclear extract. Immunoblots of the pellet fraction of indicated protein added to in vitro transcription reactions (as in B). Illustration of a proposed model of molecular interactions taking place within MED1-IDR droplets in the nuclear extract is presented to the right. (D) MED1-IDR droplets compartmentalize machinery necessary for the in vitro transcription reaction. Autoradiograph of radiolabelled RNA products of in vitro transcription reactions under indicated conditions. Arrow indicates expected RNA product. Reactions conducted as in (69) with minor modifications. See Materials and Methods for full details. Illustration of a proposed model of molecular interactions taking place within MED1-IDR droplets in nuclear extract and the impact on in vitro transcription reaction is presented to the right.