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1.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 2. The association between P/CAF and MyoD occurs only in differentiated cells. (A) Western analysis of immunoprecipitates of MyoD obtained from extracts of proliferating C2 cells and C2 cells cultured in DM for the indicated times. The probe was anti-P/CAF. The lower panel represents the level of MyoD in extracts of C2 cells cultured in DM for the indicated times, as judged by western analysis with the use of anti-MyoD. An anti-GAPDH antibody was also used to ensure equal loading. (B) The level of P/CAF in extracts of proliferating cells (GM) or cells cultured in DM for the indicated times was analyzed by western blotting using anti-P/CAF antibody as probe. The membrane was also probed with anti-GAPDH to monitor equal loading of the extracts.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.
2.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 6. HDAC1 inhibits myogenic conversion of fibroblasts to muscle cells. (A) 10T1/2 cells grown on glass coverslips were transfected in parallel with the indicated plasmids. Twenty-four hours later, the transfected cells were switched to DM, and after 48 h the cultures were fixed and immunostained with a rabbit polyclonal antibody specific for MyoD (green), a mouse monoclonal antibody specific for MHC (red) and a goat polyclonal antibody specific for HDAC1 and mutant version H141A (blue). Confocal fluorescence microscopy of random fields of cells was performed accordingly (see Materials and methods), and the left-most panels represent corresponding fields obtained by differential interference contrast (DIC) microscopy. (B) The histogram depicts the percentage of cells undergoing myogenic conversion as described in Materials and methods. The experiments were performed three separate times with comparable results.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.
3.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 4. Histone deacetylase activity associates with MyoD in undifferentiated cells, and HDAC1 alone can deacetylate MyoD in vitro. (A) Immunoprecipitates of MyoD or HDAC1 were recovered from extracts of C2 cells cultured in either GM or DM for 96 h using the indicated antibodies. The precipitates were then separately added to [14C]acetate-labeled histones, which were previously acetylated in vitro by FLAG-p300 in the presence of [14C]acetyl-CoA. The deacetylase assays were performed as described in Materials and methods, and the reaction mixtures were then separated by 18% SDS–PAGE and visualized by fluorography. The intensity of the bands on the gel was quantified by using NIH Image software, and the values were then normalized with respect to the control antibody (NR IgG). Results are expressed as a relative percentage. (B) GST–MyoD was incubated with purified FLAG-P/CAF in the presence of [14C]acetyl-CoA, captured by glutathione–agarose beads, and then separately incubated with the following: FLAG-HDAC1, FLAG-H141A or without either. Deacetylase reactions were separated by 10% SDS–PAGE, stained with Coomassie Blue (bottom panel) and then treated accordingly (see Materials and methods) for viewing by fluorography.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.
4.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 3. HDAC1 associates with MyoD in undifferentiated cells and binds directly to MyoD in vitro. (A) Immunoprecipitates of MyoD from extracts of proliferating (GM) or differentiated (DM) C2 cells were analyzed by western blotting for the presence of HDAC1, using anti-HDAC1 as probe. (B) The level of HDAC1 in extracts of proliferating C2 cells (GM) or in C2 cells switched to DM for the indicated times was analyzed by western blotting using anti-HDAC1 as probe. As a control for equal loading, the membrane was also probed with anti-GAPDH. (C) Equal molar quantities of purified GST (lane 2), GST–MyoD (lanes 3 and 5), GST–Rb pocket protein (lane 4) or mutant GST–MyoD proteins (lanes 6–11) were pre-bound to glutathione beads and then incubated with BSA for 2 h. Beads with or without attached protein (lane 1) were incubated in parallel with an equivalent amount of baculovirus-expressed, FLAG-epitope-tagged HDAC1, and after extensive washing the bound material was subject to analysis by western blotting using anti-FLAG as probe. A summary of these results is shown in the bottom panel. DM, deletion mutant; TM, truncation mutant. The basic/HLH region includes amino acids 102–166.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.
5.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 5. HDAC1 inhibits P/CAF’s ability to enhance MyoD-dependent transcription. (A and B) Proliferating 10T1/2 or C2 cells were co-transfected with the luciferase reporter vector and the indicated expression plasmids: pCMV-MyoD, pCMV-P/CAF, pCMV-HDAC1, pCMV-H141A and pCMV-β-Gal. Twenty-four hours post-transfection, the cells were switched to DM for 36 h, harvested, and assayed for reporter gene activity. Values from luciferase assays are expressed as fold activation and represent the average of three independent experiments (error bars, SD) that were normalized to β-galactosidase activity (internal standard). (C) Proliferating 10T1/2 cells were co-transfected with pCMV-β-Gal along with the indicated plasmids. After 24 h, the transfected cells were switched to differentiation medium (DM) and 48 h later the cells were harvested. Extracts from each of the transfected cells were adjusted for β-Gal activity and equivalent levels were then separated by 10% SDS–PAGE. Endogenous p21 was detected by using western blot analysis with an antibody specific for this protein. (D) The same amount of extracts as described in (C) were analyzed by western blotting for equivalent expression of β-Gal, FLAG-epitope-tagged HDAC1, H141A and MyoD, using antibodies specific for each of these proteins.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.
6.

Figure. From: A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program.

Fig. 1. Acetylation of MyoD occurs only in differentiated cells and solely by P/CAF in vitro. (A) Proliferating C2 muscle cells (GM) or C2 cells switched to DM for 24 h were pulse labeled with [3H]sodium acetate for 1.5 h. Extracts were prepared and immunoprecipitated in parallel with anti-MyoD polyclonal antibody or normal rabbit IgG (NR IgG). Immune complexes were analyzed by 10% SDS–PAGE and processed for fluorography. (B) Western analysis of proteins in nuclear extracts of proliferating C2 cells (GM) and C2 cells cultured in DM for the indicated times. Acetylated proteins were identified by anti-acetyl-lysine antibodies (lanes 1–3). Re-probing of the same blot with anti-MyoD confirmed the acetylated species to be MyoD (lanes 4–6). Note that because MyoD and the immunoglobulin heavy chain migrate extremely close to one another on SDS–polyacrylamide gels due to the similarity in molecular weight, the probing of immune complexes of MyoD from C2 cells cultured in GM or DM with an anti-acetyl-lysine antibody was problematic; and only occasionally were the results identical to those in lanes 1–3 (data not shown). It should also be noted that in these, as well as in the rest of the experiments described below, the protein content of extracts from differentiated or undifferentiated C2 cells was normalized as described in Materials and methods. (C) GST (2 µg), GST–MyoD (1 µg) or histones (5 µg) were incubated with [14C]acetyl-CoA and either wild-type P/CAF, P/CAF (Δ609–624), p300 (965–1810) or p300 (Δ1603–1653). Reaction products were separated by SDS–PAGE and visualized by fluorography. (D) Diagram of recombinant p300 and P/CAF showing the endpoints of mutant deletions in the HAT domain.

Asoke Mal, et al. EMBO J. 2001 April 2;20(7):1739-1753.

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