Journal: The Journal of biological chemistry

Article Title: Hepatitis C virus core protein binds to a DEAD box RNA helicase.

doi: 10.1074/jbc.274.22.15751

Figure Lengend Snippet: FIG. 1. Primary structures of DBX, PL10, and Ded1 and their interac- tions with HCV core protein in the yeast two-hybrid assay. A, alignment of deduced amino acid sequences of DBX (Gen- Banky accession number AF000982), PL10 (GenBanky accession number J04847), and Ded1p (GenBanky accession number X57278) is shown. Identical amino acids are shown as white on cyan. Conserved substi- tutions are shown as black on magenta. Dots represent gaps to optimize alignments, which were obtained using the Pileup pro- gram. B, two-hybrid assays showing interac- tion of HCV core protein with DBX and PL10 but not with Ded1p. Yeast strain Y190 was co-transformed with a plasmid express- ing the cytoplasmic domain of HCV fused to the GAL4 DNA binding domain and plas- mids expressing either a portion of DBX or the corresponding portions of PL10 or Ded1p fused to the GAL4 transcriptional activation domain. Transformants giving b-galactosid- ase activity (positive interactions) are blue. Control reactions of DBX, PL10, and Dep1p GAL 4 activation domain fusion proteins with GAL4 DNA binding domain alone were negative (data not shown).

Article Snippet: The amplified DNA was cloned into the GAL4 DNA binding domain fusion vector pAS2–1 (CLONTECH) to yield pAS2–1-HCV-core1–123.

Techniques: Y2H Assay, Transformation Assay, Plasmid Preparation, Binding Assay, Expressing, Activation Assay, Activity Assay, Control

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

Journal: PLoS ONE

Article Title: Rice Mitogen Activated Protein Kinase Kinase and Mitogen Activated Protein Kinase Interaction Network Revealed by In-Silico Docking and Yeast Two-Hybrid Approaches

doi: 10.1371/journal.pone.0065011

Figure Lengend Snippet: OsMKK3, OsMKK4, OsMKK6 and OsMKK10-2 were fused with GAL4 DNA-binding domain and were used as a bait against each of the fifteen rice MAPKs (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK16-2, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-2, OsMPK20-3, OsMPK20-4, OsMPK20-5 and OsMPK21-2) fused with GAL4 activation domain as preys. OsMKK1 fused with GAL4 activation domain as prey and fifteen MAPKs fused to DNA-binding domain baits were used to study protein interactions. Plasmids for bait and prey were co-transformed in AH109 yeast strain in different combinations as mentioned and selected on nutrient medium lacking Leu and Trp (upper panels). Interaction of bait and prey combinations were checked by assessing growth of co-transformed colonies streaked on selective medium lacking Ade, His, Leu and Trp (lower panels). p53 and SV40 large T-antigen are two proteins that are known to interact in yeast were used as a positive control.

Article Snippet: For initial screening MAPKKs were fused with GAL4 DNA binding domain (vector pGBKT7, BD biosciences) while MAPKs with the GAL4 activation domain (vector pGADT7, BD biosciences).

Techniques: Binding Assay, Activation Assay, Transformation Assay, Positive Control