1. Ras Induces Mediator Complex Exchange on C/EBPβ
Xianming Mo, Elisabeth Kowenz-Leutz, Hong Xu, Achim Leutz 
Molecular Cell 
Volume 13, Issue 2, Pages (January 2004)
DOI: /S (03)

2. Figure 1
C/EBPβ Interacts with Mediator and Recruits Mediator to Target Genes
(A) Left: CoIP of C/EBPβ with Mediator component CRSP130/Sur2. C/EBPβ and CRSP130/Sur2 (Flag- and HA-tagged, respectively) were cotransfected into HeLa cells, immunoprecipitated as indicated, separated by SDS-PAGE, blotted, and examined by immunostaining (top panel, anti-HA; lower panel, anti-Flag). Right: Input control represents 10% of the material used for IP as shown on the left.
(B) C/EBPβ recruits the Mediator complex to promoters of target genes. Schematic representations of mim-1 and lysozyme genes indicating the location of the amplification products (gray bars), TATA boxes, transcription start sites (+1), and translation stop sites. QT6 cells were transfected with C/EBPβ-Flag or vector control. Cell lysates were analyzed by ChIP with anti-Flag antibody, anti-CDK8 antibody, or a mixture of control mouse and goat serum as indicated. Coprecipitated DNA was analyzed by PCR amplification. A negative image of an ethidium bromide-stained gel is shown. Input lanes represent 0.1% of material used for IP. The asterisk near the bottom panel indicates an unspecific amplified product that conveniently serves as input control.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 C/EBPβ Interacts with the Mediator Component CRSP130/Sur2
(A) Schematic representations of C/EBPβ proteins indicating the conserved domains. HeLa cells were transfected with C/EBPβ-Flag constructs as indicated. Cell lysates were subjected to CoIP with antibodies to CDK8, CRSP130, CRSP150, or goat serum as a control. Coprecipitated proteins were revealed with anti-Flag antibody. Input (right panel) represents 10% of the material used for IP.
(B) Mapping of C/EBPβ fragments that interact with CRSP130/Sur2. Left: Schematic representation of C/EBPβ segments fused to GST. Matrix-bound GST fusion proteins or GST controls were incubated with in vitro-translated, 35S-labeled methionine CRSP130/Sur2 protein. Specifically bound proteins were subjected to SDS-PAGE and revealed by fluorography (upper panels). Bottom panels (GST protein) show expression of GST constructs used in the binding experiments as shown above. Input represents 15% of the material used for pull-down assays.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
The Interaction between C/EBPβ and Mediator Requires CRSP130/Sur2 in Live Cells
(A) Flag-tagged C/EBPβ was coexpressed with HA-tagged E1ACR3VP16 or HA-tagged E1ACR3R177KVP16 in HeLa cells as indicated. Cell lysates were subjected to CoIP with anti-CDK8 antibody, electrophoresis, and protein blotting. CoIP of C/EBPβ was determined with anti-Flag antibody. Input represents 10% of material used for IP.
(B) Small hairpin RNA knockdown of CRSP130/Sur2 expression. NIH 3T3L1 fibroblasts transfected with 10 μg of pSuper shRNA vectors encoding shRNAC1 or shRNAC2 and 1 μg of pBabe-puro plasmid were selected with 2.5 μg/ml puromycin, expanded, and analyzed for CRSP130/Sur2 protein expression. CDK8 protein expression serves as specificity control for the shRNA construct.
(C) Elimination of CRSP130/Sur2 protein abrogates association of C/EBPβ with Mediator. Flag-tagged C/EBPβ was transiently transfected into NIH 3T3-L1 carrying the indicated siRNA or control vectors. Cell lysates were subjected to IP using CDK8 antibody. Coprecipitated C/EBPβ was revealed by immunoblotting using anti-Flag antibody. Input represents 10% of the material used for IP.
(D) Reconstitution of Mediator binding in CRSP130/Sur2 knockdown cells by human CRSP130. Different amounts of HA-tagged human CRSP130/Sur2 (1, 2, 4 μg plasmid) were cotransfected into CRSP130/Sur2 knockdown NIH 3T3L1 cells. IP using CDK8 antibody was performed 48 hr later. CoIP of C/EBPβ was examined by immunoblotting using anti-Flag antibody. Input represents 10% of material used for IP.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Functional Significance of C/EBPβ-CRSP130/Sur2-Bound Mediator
C/EBPβ or Gal4VP16 were cotransfected with siRNA vectors into NIH 3T3-L1 fibroblasts together with the −85cMGF-luciferase reporter (open bars) or Gal4-luciferase reporter (closed bars) as indicated. Forty-eight hours after transfection, cell extacts were prepared and analyzed for reporter activity.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Active and Inactive Forms of C/EBPβ Bind to Different Mediator Complexes
(A) Flag-tagged full-length C/EBPβ, activated C/EBPβ (ΔCR57 C/EBPβ), or transcriptionally inactive C/EBPβ (ΔCR1234 C/EBPβ) were transfected into HeLa cells. Cell lysates were immunoprecipitated with anti-CRSP70 or anti-CDK8 antibody, separated by SDS-PAGE, and blotted. CoIP of C/EBPβ constructs was revealed with anti-Flag antibody.
(B) Flag-tagged C/EBPβ and EJ-Ras or control vector were cotransfected into HeLa cells. Cell lysates were subjected to IP with antibodies directed against CDK8, CRSP70, CRSP130, as indicated on the top, separated by SDS-PAGE, and blotted. CoIP of C/EBPβ was revealed with anti-Flag antibody.
(C) Flag-tagged, activated ΔCR7 C/EBPβ or C/EBPβ218E mutant was transfected into HeLa cells. Cell lysates were subjected to IP with antibodies to CDK8, CRSP70, and CRSP130. CoIP of C/EBPβ constructs was revealed with anti-Flag antibody. Input represents 10% of the material used for IP.
(D) Flag-tagged C/EBPβ and EJ-Ras or control vector were cotransfected into HeLa cells. Cell lysates were subjected to IP with anti-Flag antibody and blotting following electrophoresis. CoIP of PolII was revealed with 8WG16 antibody. Input represents 10% of the material used for IP.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Repressive and Actived C/EBPβ Recruits Different Mediator Complexes
(A) HeLa cells were transfected with EJ-RAS constructs as indicated. Cell lysates were subjected to CoIP with antibodies to CDK8, CRSP70 as indicated. Coprecipitated proteins were revealed with anti-C/EBPβ antibody (arrowheads). Input represents 10% of the material used for IP.
(B) EJ-Ras or dominant-negative dn-Ras were transfected into HeLa cells together with the −85cMGF-luciferase reporter, HSP70.2, or Gal4-luciferase reporter as indicated. Reporter activity was analyzed after 48 hr.
(C) Repressive and activated C/EBPβ recruit different Mediator complexes to target promoters. Schematic representations of cMGF promoter construct and HSP70.2 genes indicating location of amplification products (gray bars), TATA boxes, transcription start sites (+1), and translation stop sites. HeLa cells were transfected with EJ-Ras, dn-Ras, or vector control. Cell lysates were analyzed by ChIP with anti-C/EBPβ antibody, anti-CDK8 antibody, anti-CRSP70 antibody, or a mixture of control mouse and goat serum, as indicated. Coprecipitated DNA was analyzed by PCR amplification. A negative image of an ethidium bromide-stained gel is shown. Input lanes represent 0.1% of material used for IP. The asterisk near the first right panel indicates an unspecific amplified product that serves as input control.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Differential Binding of Activated and Repressed C/EBPβ to CRSP130/Sur2
(A) Binding of CRSP130/Sur2 fragments F1 to F5 to the transactivation and repression domains of C/EBPβ. Top: Schematic representation of CRSP130/Sur2 fragments. Pull-down: Matrix-bound GST-CR1234 or GST-CR567 proteins were incubated with in vitro-translated, 35S-labeled methionine fragments of CRSP130/Sur2 (F1 to F5). Interacting proteins were revealed by SDS-PAGE and fluorography. Expression controls are shown on the right. Input represents 15% of the material used for pull-down assays.
(B) CoIP of C/EBPβ constructs with fragments of CRSP130/Sur2. Wt C/EBPβ, C/EBPβΔCR6, or C/EBPβΔCR7 and the HA-tagged full-length CRSP130/Sur2 or F1 and F4 constructs from Figure 7A, respectively, were cotransfected into HeLa cells, immunoprecipitated with HA-antibody, separated by SDS-PAGE, blotted, and examined by anti-C/EBPβ immunostaining. Bottom: Input control represents 10% of the material used for IP.
Molecular Cell  , DOI: ( /S (03) )