E2F6 binds to both amino and carboxy terminal domains of EBNA3C.
A) Myc-tagged EBNA3C full length and different truncated mutants were translated in vitro using a T7-TNT kit. The S35-radiolabeled in vitro-translated proteins were pre-cleared with GST-beads, then incubated with GST control or GST-E2F6 protein and GST beads. The mixture were resolved by 10% SDS-PAGE, exposed to phosphorImager plates and scanned with Typhoon Scanner. B-C) Cell lysates form 50 million different B-cells (BJAB, BJAB7, BJAB10, LCL1, LCL2) were collected and incubated with either GST control or GST-E2F6 protein and GST beads. The mixture samples were identified by western blot analysis using specific mouse antibody for EBNA3C (A10). D) Purified GST control and GST-E2F6 proteins were separated by 10% SDS-PAGE and stained with Coomassie Blue. E) The schematic diagram shows various interactive domains of EBNA3C with E2F6.