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. 2022 Feb 12;50(5):2566–2586. doi: 10.1093/nar/gkac081

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Regulation of MEF2-class IIa HDACs axis by PAOA-derivatives. (A) GST pull-down assay, using recombinant MEF2D (1–190) or GST as control. Purified GST or GST-MEF2D recombinant proteins (2 μg) were incubated with cellular lysates obtained from NIH3T3 cells overexpressing HDAC4 mutated in 14–3–3 binding sites. Two different concentrations [14 and 42 μM] were used. Immunoblots were performed to visualize HDAC4 or recombinant GST. (B) SK-UT-1 cells were treated for the indicated times with NKL54 [5 μM] or SAHA [2.5 μM]. Cellular lysates were generated and immunoblot performed using the indicated antibodies. Actin and Histone 3 (H3) were used as loading control. (C) Lysates from SK-UT-1 cells treated for 12 h with DMSO or with SAHA [2.5 μM] or NKL54 [5μM] MEF2D-HDAC4 complexes were immunoprecipitated with antibodies against HDAC4 or USP33, as a control. Immunoblotting using an anti-MEF2D antibody was next used for the detection of the MEF2-HDAC4 complexes. Asterisks point to IGs.