1. Volume 26, Issue 5, Pages 717-729 (June 2007)
Purification of a Plant Mediator from Arabidopsis thaliana Identifies PFT1 as the Med25 Subunit 
Stefan Bäckström, Nils Elfving, Robert Nilsson, Gunnar Wingsle, Stefan Björklund 
Molecular Cell 
Volume 26, Issue 5, Pages (June 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Isolation of Mediator from A. thaliana Whole-Cell Protein Extracts
(A) Purification scheme. The horizontal lines indicate stepwise elution with potassium acetate concentrations indicated in mM.
(B) Immunoblot analysis of Biorex-70 fractions with monoclonal antibody 8WG16 (to reveal the largest Pol II subunit), and with 1 μg/ml and 3 μg/ml of affinity-purified anti-AtMed6 and anti-AtMed7.
(C) Same as in (B), but on fractions from the DEAE-Sephacel column.
(D) Immunoprecipitation by affinity-purified anti-AtMed6 antibody coupled to protein A-Sepharose. A fraction from the 200 mM potassium acetate eluate from the DEAE-Sephacel column (Load, 150 μg) was incubated with affinity-purified anti-AtMed6 antibody beads. Equal amounts of protein from the load and supernatant (Sup) and six times more protein from the pellet (Pel) were analyzed by immunoblotting with antibodies against the proteins indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Identification of the A. thaliana Mediator Subunits
(A) 1D gel electrophoresis of A. thaliana Mediator. Eluted protein from the immunoprecipitation described in Figure 1D was subjected to SDS-PAGE (12%) and visualized by colloidal Coomassie staining (lane 1). As a reference, lane 2 contains the same as lane 1, except that no 200 mM potassium acetate eluate from the DEAE-Sephacel column was added. The molecular masses of standard proteins are indicated to the right (lane M). Numbers to the left indicate protein-containing gel slices subjected to in-gel tryptic digestion and LC-ESI-MS/MS.
(B) 2D gel electrophoresis of A. thaliana Mediator. Eluted protein from the immunoprecipitation described in Figure 1D was separated in the first dimension by isoelectric focusing followed by separation on SDS-PAGE (12%). Estimates of molecular mass (kDa) and pI are indicated on the ordinate and abscissa, respectively. Immobilized pH gradient was pH 3–10. Numbers outside the circles indicate spots subjected to in-gel tryptic digestion and LC-ESI-MS/MS (See Table 2).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Identification of PFT1 as the A. thaliana Med25 Subunit
(A) Mediator homolog sequences from A. thaliana, O. sativa (gi: ), H. sapiens (gi: ) and D. melanogaster (gi: ) were aligned using T-coffee and colored with TeXshade. Red-shaded background indicates identical amino acids, orange indicates amino acids conserved in three out of four species, and yellow indicates similar amino acids in three out of four species. Amino acids considered similar were F/Y/W, I/L/V/M, R/K, D/E, G/A, S/T, and N/Q. The black boxes indicate a region corresponding to the mapped Mediator-interacting von Willebrand factor type A (VWA) domain of human Med25 (amino acids 1–226). The blue boxes indicate a region corresponding to the minimal VP16 interaction domain of human Med25 composed from the results presented by Mittler et al. (2003) and Yang et al. (2004).
(B) Sequence alignments of Med25 from the plants A. thaliana, P. trichocarpa ( O. sativa, and P. patens ( The black and blue boxes indicate the regions in the plant Mediator sequences that correspond to the same regions in the human Med25 sequence as indicated in (A).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Identification of At1g11760 as an A. thaliana Mediator Subunit
(A) Immunoblot analysis of Biorex-70 fractions (as in Figure 1B) with affinity-purified antibodies raised against a C-terminal peptide from At1g11760.
(B) Immunoprecipitation by the anti-AtMed6 antibody. A fraction from the 200 mM potassium acetate eluate from the DEAE-Sephacel column was incubated with affinity-purified anti-AtMed6 antibody beads as described in Figure 1D.
(C) Immunoprecipitation from the 200 mM potassium acetate eluate from the DEAE-Sephacel column. Protein from this fraction (120 μg for each immunoprecipitation) was incubated with antibody beads. The supernatant was removed, the beads were washed, and immunoprecipitated protein was eluted. Protein from the load (Load) and each of the anti-AtMed6, anti-At1g11760, and anti-GST immunoprecipitation pellets was analyzed by immunoblotting with antibodies against the proteins indicated to the right. Six times more protein from the pellets was used in both (B) and (C).
(D) Venn diagrams illustrating the overlap between conserved Mediator proteins (left) and nonconserved (right) identified in the immunoprecipitation using anti-AtMed6 (solid lines) or anti-At1g11760 (dashed lines) antibody beads. Protein hits that are considered paralogs are only counted once.
(E) Summary of common and species-specific Mediator subunits in S. cerevisiae, H. sapiens, and A. thaliana. Gray fields indicate conserved Mediator subunits, white fields represent subunits that are absent in Mediator from each species, and black fields represent species-specific subunits. Accession numbers to the different A. thaliana Mediator subunits are indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
A Proposed Model for AtMed25/PFT1 Function in the Light-Quality Pathway that Regulates Flowering Time
The red to far red (R/FR) ratio of incoming light is sensed by phyB in leaves. This signal is transmitted to the A. thaliana Mediator complex, either by direct action on the AtMed25 subunit or indirectly via promoter-bound transcriptional regulatory proteins to affect transcription of target genes, here represented by FT.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions