Journal: Nucleic Acids Research

Article Title: The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members

doi: 10.1093/nar/gkt199

Figure Lengend Snippet: ERM binds to MED25. ( A ) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. ( B ) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro . GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. ( C ) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro . GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. ( D ) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP α Flag) and immunoblotted with anti-Flag (IB α Flag) and anti-ERM (IB α ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). ( E ) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB α MED25) or polyclonal anti-ERM (IB α ERM).

Article Snippet: The ERM activation domain derivatives (1–72, 1–38, 38–72), E1A 13S and E2F were subcloned into pBIND (Gal4 DNA-binding domain) vector (Promega).

Techniques: Binding Assay, In Vitro, Generated, Autoradiography, Western Blot, SDS-Gel, Staining, Expressing, Incubation, Produced, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members

doi: 10.1093/nar/gkt199

Figure Lengend Snippet: Effects of MED25 on transcriptional activation by the ERM TAD. ( A–D ) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ΔACID, ΔCt, ΔNt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ΔACID or the expression of MED25 WT, MED25 ΔCt, MED25 ΔNt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.

Article Snippet: The ERM activation domain derivatives (1–72, 1–38, 38–72), E1A 13S and E2F were subcloned into pBIND (Gal4 DNA-binding domain) vector (Promega).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Sequencing, Western Blot