1. The TBN Protein, which Is Essential for Early Embryonic Mouse Development, Is an Inducible TAFII Implicated In Adipogenesis 
Mohamed Guermah, Kai Ge, Cheng-Ming Chiang, Robert G Roeder 
Molecular Cell 
Volume 12, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
Amino Acid Sequence Comparisons of hTAFII43 and Related Proteins
(A) hTAFII43. The amino acid sequences obtained by microsequencing of hTAFII43 are underlined. The first underlined sequence was used to derive degenerate oligonucleotides for screening.
(B) Human histone H4 versus hTAFII43.
(C) Alignment of Drosophila PRODOS, mouse TBN and hTAFII43 sequences. The complete hTAFII43 (TAF8) cDNA sequence has been deposited in the GenBank database of NCBI with the accession number AF
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Polypeptide Composition of TFIID Purified through FLAG-Tagged hTAF8 (f:43) and FLAG-Tagged TBP Subunits
(A) TFIID purified from f:43 cells. The complex was resolved by SDS-PAGE and visualized by silver staining.
(B) Comparison of TFIIDs purified from f:TBP and f:43 cell lines. As indicated, increasing amounts (in μl) of purified TFIID from the f:TBP cell line and 10 μl of purified TFIID from the f:43 cell line (f:43) or from HeLa nuclear extract (NE) were resolved by SDS-PAGE and analyzed by Western blotting with antibodies generated against the indicated TFIID subunits or the RPB1 and SRB11 control proteins.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 TAF8 Is an Integral Subunit of a Functional TFIID
(A–C) TFIID fully occupied by TAF8 mediates both basal and activator-dependent transcription. Transcription was assayed in a purified transcription system containing TFIID prepared from either the f:43 or the f:TBP cell line. All reactions with TFIID contained the equivalent of 4 ng of TBP. (A) The standard TFIID-dependent transcription assay was supplemented with TFIID from the f:TBP cell line (lanes 3 and 4) and with 20 ng of purified fGAL4-p65 (lanes 2 and 4) as indicated. The test template consisted of a GAL4-site containing promoter (pG5E1b) and the control template consisted of pMLΔ53 (C).
(B) Assays were equivalent to those in (A) but contained either TFIID from the f:TBP cell line (lanes 1 and 2) or TFIID from the f:43 cell line (lanes 3 and 4). C, Assays contained either TFIID from the f:TBP cell line (lanes 1 and 2) or TFIID from the f:43 cell line (lanes 3 and 4) and Sp1 (lanes 2 and 4) as indicted. The test template consisted of an Sp1-responsive template (HIV-1, pMHIVWT) (Guermah et al., 1998) and the control template consisted of pMLΔ53.
(D) hTAF8 is associated predominately with TFIID. Untreated HeLa nuclear extract (NE) or HeLa nuclear extract (NEΔD) immunodepeleted by incubation, at high stringency, with anti-TBP and anti-TAF5 antibodies was subjected to Western blot analysis. Increasing amounts (in μl) of nuclear extracts were analyzed, as indicated, and TFIID subunits corresponding to the antibodies used are indicated.
(E and F) hTAF8 interacts with several other TFIID subunits. (E) In vitro interactions between the hTAF8 and highly purified TFIID subunits. TBP and TAF8 were immunoprecipitated by either anti-hTAF8 antibodies (αTAF8 IP) or control antibodies (control IP) following incubation with hTAF8 protein and washing. Immunoprecipitates and 10% of each input were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated TFIID subunits. (F) Specific in vitro interaction between TAF10 and hTAF8. Purified hTAF10 was incubated, in high salt buffer (300 KCl) containing 0.1% NP-40 and 0.2 mg/ml of bovine serum albumin, with equivalent amounts of either GST (glutathione S-transferase) alone or a GST-hTAF8 fusion protein immobilized on glutathione-Sepharose beads. After extensive washing, bound proteins were resolved by SDS-PAGE and assayed by Western blot. The input sample contained 10% of the amount used for binding. (G) hTAF8 interacts with hTAF10 mainly through its histone fold (HF) domain (residues 1-101) and not through its C-terminal portion (Delta HF) (residues ). Experiments were performed as in (F).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Selective Induction of hTAF8 and Incorporation into TFIID during Adipogenesis
(A) Western blot analysis of whole-cell extract (WCE) proteins derived from preadipocytes and differentiated adipocytes, as well as C2C12 myoblasts and differentiated C2C12 cells (C2C12 diff.), using antibodies to the indicated TAFs, to PPARγ (marker of adipogenesis), and to myogenin (marker of myogenesis).
(B) Nuclear extract from adipocytes was immunodepleted, at high stringency, of TBP and TAFs (NEΔD). Equivalent amounts of nondepleted and depleted extracts were analyzed by Western blot using antibodies to the indicated TFIID subunits.
(C) Western blot analysis of whole cell-extract proteins derived from NIH-3T3 cells expressing PPARγ (NIH/γ) before differentiation (D0) and after 8 days of differentiation (D8) in the presence (+Pio) or absence (-Pio) of pioglitazone. Antibodies used are to the indicated proteins.
(D) Morphological changes of preadipocytes and C2C12 cells upon differentiation.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
The TAF8 Histone Fold (HF) Acts as a Specific Dominant-Negative Mutant of 3T3-L1 Differentiation In Vitro
(A–C) 3T3-L1 cells were infected with either control retroviruses (vector alone) or with recombinant retroviruses expressing different hTAF8 derivatives. Upon puromycin selection, cells were induced to differentiate and subjected to Oil Red O staining. The TAF8 HF inhibits the morphological differentiation of 3T3-L1 cells.
(D) Ectopic expression of hTAF8 and its derivatives has no effect on the morphological differentiation of C2C12 cells in vitro.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
hTAF8 Acts as a Positive Regulator of 3T3-L1 Differentiation and Reverses the Inhibitory Effect of Its HF Domain
(A)The TAF6 HF domain is expressed in 3T3-L1 cells and gets incorporated into the TFIID complex. Western blot of TAF6 HF expression in 3T3-L1 cells after immunoprecipitation (IP) by the FLAG antibody. The TAF6 HF derivative carries a FLAG tag at its N terminus. Coimmunoprecipitation of full-length TAF5 and TAF9 indicates that the TAF6 HF domain is incorporated into TFIID.
(B) A graph showing that ectopically expressed TAF6 and TAF9 HF domains have no significant effect on 3T3-L1 differentiation. Control 3T3-L1 cells and 3T3-L1 cells expressing TAF6 HF or TAF9 HF were induced to differentiate and triglyceride accumulation was quantitated.
(C) Western blot of whole-cell extracts from 3T3-L1/TAF8 HF cells expressing vector (hygromycin) or full-length hTAF8. An anti-TAF8 antibody was used as a probe.
(D) A graph showing that ectopic expression of the full-length TAF8 reverses the inhibitory effect of its HF on 3T3-L1 cell differentiation. 3T3-L1/TAF8 HF cells were infected either with vector control (hygromycin) or with a TAF8-expressing pMSCV-hygromycin vector. The experiment was performed as in (B).
(E) The ectopically expressed full-length TAF8 in 3T3-L1 cells is incorporated into the TFIID complex. Western blot analysis of TAF8 ectopically expressed in 3T3-L1 cells after immunoprecipitation (IP) by the anti FLAG antibodies. The TAF8 carries a FLAG tag at its N terminus. Coimmunoprecipitation of full-length TAF4, TAF5, and TAF9 indicates that TAF8 is incorporated in TFIID.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Ectopic Expression of the hTAF8 HF Domain Specifically Downregulates Expression of C/EBPα and PPARγ, as well as GLUT4 and IRβ Proteins, upon Differentiation of 3T3-L1 Cells
(A) Western blot of whole-cell extracts from 3T3-L1 cells expressing vector control, FLAG-hTAF8, and FLAG-Delta HF and an anti-FLAG immunoprecipitate of induced cells expressing the FLAG-TAF8 HF domain. The probe was an antibody raised against residues of TAF8. Since these residues (and corresponding epitopes) are differentially present in TAF8 (1-310), Delta HF ( ) and HF (1-101), the antibody does not uniformly recognize TAF8 and its derivatives.
(B) Western blot analysis of whole cell-extract proteins derived from differentiated 3T3-L1 cells expressing the vector control or hTAF8, Delta HF, and HF proteins. Antibodies to the indicated proteins were employed.
(C) Western blot of whole cell-extract proteins derived from differentiated C2C12 cells expressing the vector control, hTAF8, Delta HF, and HF proteins. Antibodies used are to the myogenin.
Molecular Cell  , DOI: ( /S (03) )