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. 2017 Dec 29;15(3):3147–3154. doi: 10.3892/ol.2017.7718

Figure 1.

Figure 1.

MED28 is a ZNF224-binding protein. (A) Protein structure of ZNF224/ZNF255 and mediator complex subunit 28 (MED28). ZNF224 has conserved KRAB (aa 8–67) and zinc finger (aa 190–641) domains. MED28 has a PRD (aa 13–39) and MED domain (aa 72–178), which is shared between the paralogous mediator complex subunit proteins. Myc-MED28 was transfected into 293 cells in the presence of Flag-ZNF224 or Flag-ZNF255 for 24 h. Cell extracts were subjected to an immunoprecipitation assay using (B) anti-Myc or (C) anti-Flag antibodies; samples were separated by SDS-PAGE with WCL. Immunoblotting was performed with the indicated antibodies, and γ-tubulin was used as a loading control. (D) Myc-MED28 was transfected into 293 cells in the presence of Flag-ZNF224, Flag-ZNF255, or HA-ZNF224-N (aa 1–67). Cell lysates were precipitated using an anti-Myc antibody and immunoblotted with the indicated antibodies. (E) Ponceau-S stain showing purified GST (lane 2), GST-MED28 full length (lane 3), aa 1–71 (lane 4), aa 1–151 (lane 5), aa 43–176 (lane 6), aa 61–176 (lane 7), and aa 72–176 (MED domain, lane 8). Cell lysates from Flag-ZNF224-transfected 293 cells were incubated with GST fusion proteins for 4 h, and samples were analyzed using SDS-PAGE and immunoblotted with an anti-Flag antibody. MED28, mediator complex subunit 28; ZNF224, zinc finger protein 224; aa, amino acid; KRAB, Krüppel-associated box; PRD, proline rich domain; HA, hemagglutinin; Myc, Myc proto-oncogene protein; WCL, whole cell lysates; GST, glutathione S-transferase.