Skip to main content
. 2018 Dec 1;32(23-24):1537–1549. doi: 10.1101/gad.318436.118

Figure 4.

Figure 4.

HCF1 and BAP1 are new components of the DRED complex and regulate NCoR1 activity in the β-globin locus. (A) Immunoprecipitated HCF1 was interrogated on Western blots using antibodies recognizing HCF-1, TR4, NCoR1, or BAP-1 in wild-type HUDEP-2 cells. (B) BAP1 knockdown enhances nuclear protein ubiquitination with virtually no change in NCoR1 abundance in 293T cells. (C) shRNA knockdown of BAP1 modestly increased NCoR1 ubiquitination in 293T cells. 293T nuclear extraction from wild type (lane 1), HA-ubiquitin expression plasmid (lane 2), or the combination of HA-ubiquitin and shBAP1 plasmids (lane 3) was immunoprecipitated with anti-NCoR1 or anti-HA followed by immune blotting with anti-HA or anti-NCoR1 antibodies. (D) BAP1 shRNA knockdown in HUDEP-2 cells affects NCoR1 and HCF1 protein abundance and slightly reduces LSD1 and LRF protein levels but significantly reduces MYB protein abundance. (E) NCoR1 mRNA level increased by only 1.7-fold after BAP1 knockdown. (F) NCoR1 occupancy at the TR2/4-binding site in HS2 is significantly reduced after BAP1 knockdown in undifferentiated HUDEP-2 cells, and the reduced NCoR1 occupancy could be partially rescued by proteasome inhibitor lactacystin treatment. (Blue bar) Control scrambled shRNA-infected HUDEP-2 with IgG control; (red bar) control HUDEP-2 cells with anti-NCoR1; (green bar) BAP1 knockdown HUDEP-2 with IgG control; (purple bar) BAP1 knockdown HUDEP-2 cells with anti-NCoR1; (gray bar) BAP1 knockdown HUDEP-2 after 3 h of treatment with 25 µM lactacystin (IgG control); (orange bar) NCoR1 detection in BAP1 knockdown HUDEP-2 cells after 3 h of treatment with 25 µM lactacystin. Data are shown as the means ± SD from three independent experiments (*) P < 0.05; (***) P < 0.001, unpaired Student's t-test.