Figure 4. Brd4‐BD2 binds acetylated‐EED to repress Foxp3 and Fbxw7 in Th2 cells.

1. Immunoprecipitation of Flag‐tagged Brd4, followed by Western blotting of HA to detect HA‐Ezh2, HA‐EED, and HA‐Suz12 in HEK293T cells transfected with Flag‐Brd4 and/or HA‐Ezh2, HA‐EED, and HA‐Suz12 plasmids.
2. HEK293T cells are transfected with Flag‐Brd4 and HA‐EED, HA‐Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag‐tagged Brd4, treated in vitro with JQ1 (5 μM), MS402 (5 μM), and ABBV‐744 (5 μM), followed by Western blotting of HA.
3. Th2 cells were differentiated for 6 days. Lysates are immunoprecipitated with Brd4, treated in‐vitro with ABBV‐744 (5 μM), followed by Western blotting of EED, Ezh2, and Suz12.
4. HEK293T cells are transfected with Flag‐Brd4 and HA‐EED plasmids. Lysates are immunoprecipitated with HA, followed by Western blotting of HA and pan‐acetylation (pan‐Ac). * denotes specific bands.
5. HEK293T cells are transfected with Flag‐Brd4 and HA‐EED plasmids. Lysates are immunoprecipitated with Flag‐tagged Brd4, treated in vitro with 10 μM acetylated EED peptides (K19, K211, K250, K261) and nonacetylated EED‐K19 peptide, followed by Western blotting of HA to detect HA‐EED.
6. HEK293T cells are transfected with Flag‐Brd4, HA‐EED, and HA‐EED(K19R) plasmids. Lysates are immunoprecipitated with Flag followed by Western blotting of HA, and vice versa.
7. Dual‐luciferase reporter assay of HEK293T cells transfected with Fbxw7‐promoter, Flag‐Brd4, HA‐EED, and HA‐EED(K19R) plasmids.

Data information: All Western blotting data are representative of three independent experiments. All data represent mean ± SD and are representative of three independent experiments. Data are analyzed by Paired t test. *P < 0.05.

Source data are available online for this figure.