Journal: The Journal of Biological Chemistry

Article Title: Transcription suppression is mediated by the HDAC1–Sin3 complex in Xenopus nucleoplasmic extract

doi: 10.1016/j.jbc.2022.102578

Figure Lengend Snippet: Romidepsin stimulates BRD4-dependent transcription. A , plasmid pull-down schematic. B , pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. At the indicated time points, DNA-bound proteins were isolated by plasmid pull down and visualized by Western blot with the indicated antibodies (n = 2). Input represents 3% of total reaction sample. Acetylation of H4K5 and H4K8 in total reaction samples is shown in Fig. S3 A . C , pActin was incubated in NPE supplemented with buffer or 100 μM Romidepsin. Samples were withdrawn at the indicated time points and binding of BRD4 to the actb promoter region was analyzed by ChIP (n = 3). D , pActin was incubated in NPE supplemented with Romidepsin and/or JQ1, as indicated. RNA was isolated and quantified after 120 min (n = 2). Student t test: p -value < 0.05 (∗), p -value < 0.01 (∗∗), p -value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. BRD, bromodomain; NPE, nucleoplasmic extract.

Article Snippet: Antibodies for Xenopus HDAC1, HDAC2, and BRD4 were produced by New England Peptide (NEP) using the following antigen sequences: HDAC1, KTDSKRVKEETKS; HDAC2, MDTKGVKSEQPINP; and BRD4, NFQSELMEIFEQNLFS.

Techniques: Plasmid Preparation, Incubation, Isolation, Western Blot, Binding Assay