Extended Data Fig. 1: Preparation of the human Mediator–PIC complex.
From: Structure of the human Mediator–RNA polymerase II pre-initiation complex
a, Size-exclusion chromatography of recombinant human Mediator shows a single peak. SDS–PAGE analysis (replicated three times) shows the presence of 20 Mediator subunits, confirmed by mass spectrometry (not shown). Raw SDS–PAGE data are provided in Supplementary Fig. 2. b, SDS–PAGE analysis of the human Mediator–PIC complex after isolation from a sucrose gradient (replicated three times). Raw SDS–PAGE data are provided in Supplementary Fig. 2. c, Recombinant 20-subunit human Mediator is able to stimulate Pol II CTD phosphorylation by CDK7 in the presence of ATP. Pol II CTD phosphorylation was assessed by western blotting against phosphorylated Ser5 of the CTD heptad repeat. An antibody against RPB3 was used to obtain the loading control. Experiments were performed in triplicate (P1–P3 and M1–M3) and a negative control sample without ATP (PC and MC) was included to exclude prior CTD phosphorylation. The bar diagram illustrates an around 4.5-fold stimulation of Pol II CTD phosphorylation in the Mediator–PIC samples over the PIC samples. Data are mean ± s.d. of three independent experiments (replicated three times). The mean value of the triplicates was used as the centre measure for error bars (mean = 4.47). Statistical significance with a P value of 3.43 × 10−6 was determined using a one-tailed unpaired t-test (***P < 0.001, **P < 0.01, *P < 0.05). Raw data for the western blots are available in Supplementary Fig. 2.
