Fig. 2.

Validation of interactions with human Brd4. (A) The indicated Brd4 fragments stably expressed in 293T cells were subjected to small-scale HA IPs. Equal amounts of input (IN) samples along with 50% of IPs were separated by SDS-PAGE and immunoblotted with antibodies specific to HA, ATAD5, NSD3, JMJD6, CHD4, and actin. (B) 293T cells were transiently transfected with Flag-GLTSCR1, Flag-ATAD5, Flag-NSD3, Flag-JMJD6, or Flag-CHD4, and cell lysates were harvested 48 h posttransfection. Proteins were immunoprecipitated with anti-Flag antibody and visualized as described in panel A with antibodies to Flag epitope, Brd4, or actin. (C) Endogenous IPs were performed using whole-cell lysates from 293T cells with antibodies to CHD4 or JMJD6. Bound proteins were captured using protein A beads, eluted, and analyzed by Western blotting with anti-CHD4, anti-JMJD6, anti-Brd4, or actin.