Fig 2.

In vitro interaction between KDM3A and LANA. (A) Flag-KDM3A and Flag-LANA proteins were purified from baculovirus-infected Sf9 cells. Purified proteins were subjected to SDS-PAGE and stained with Coomassie brilliant blue. (B) Schematic diagram of the domains of LANA. Deletion proteins used in this study are depicted in the middle. GST-LANA proteins were subjected to SDS-PAGE and stained with Coomassie (bottom). An in vitro GST pulldown assay was performed by incubation of purified full-length Flag-KDM3A with immobilized LANA-GST deletion mutants in binding buffer. LANA residues 191 to 251 precipitated nearly 25% of the input of full-length KDM3A. (C) Schematic diagram of the KDM3A domains based on the SMART database. Successive N-terminal GST deletion mutants of KDM3A, prepared from bacterial cells, were incubated with full-length baculovirus-purified Flag-LANA for a series of in vitro GST pulldown assays. The interaction was probed with anti-Flag antibody, and GST proteins used in these studies were visualized by Coomassie staining. (D) Confirmation of the interacting domain in HEK 293T cells. The indicated plasmids were cotransfected into HEK 293T cells. The coprecipitated deletion mutant of KDM3A was probed with anti-GFP antibody. Whole-cell lysates (WCL) were similarly probed and used as input controls.