Fig 1: MED25 mediates the interaction between ERM and Mediator. (A) Depletion of endogenous MED25 from DAMI cell nuclear extract using Halo-Tag fusion protein ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L. After incubation and extensive washing, MED25 not associated with ERM 1–72 present in the supernatant was saved as the unbound fraction (U). Samples were separated by SDS-PAGE, and MED25 was detected by immunoblotting with anti-MED25 antibody. I: Input, 10% of the nuclear extracts used in binding reactions. U: Unbound, 10%. (B) MED25 and Mediator (here documented with MED1 and MED6) bind specifically to ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L as in Figure 3A. ERM 1–72-bound Mediator complex (B) was eluted by boiling the beads in SDS buffer and was resolved by 12% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10% of the nuclear extracts used in binding reactions. B: bound material, 100%. (C) MED25 depletion of nuclear extract impairs recruitment of Mediator to ERM. Nuclear extracts from DAMI cells were sequentially incubated with Halo-Tag ERM 1–72 as indicated. After incubation and extensive washing, the supernatant (containing unbound material, U1, U2 and U3) and the affinity resin (containing bound material, B1, B2 and B3) were boiled in SDS buffer and resolved by SDS-PAGE. Specifically bound and unbound proteins were detected by western blot using the specified antibodies. I: input, 10%. U: unbound, 10%. B: bound, 100%. (D) MED25 immunodepletion reduces interaction between ERM and Mediator. Anti-MED25 antibody and non-specific anti-serum (control) were first coupled to Pureproteome protein A magnetic beads (Millipore). Nuclear extracts from DAMI cells were then incubated at 4°C with antibody-bound protein A beads. The resulting supernatant was incubated with immobilized Halo-Tag ERM 1–72 as indicated. ID: immunodepletion. Bound (B) and unbound (U) materials were resolved by 10% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10%. U: unbound, 10% and B: bound, 100%.