Fig 1.

KDM3A interacts with KSHV LANA in vivo. (A) Generation of stably 3×Flag-LANA-expressing HeLa cells. Immunoblotting was performed with anti-Flag antibody to confirm LANA expression. Cellular GAPDH was probed with anti-GAPDH monoclonal antibody and used as a loading control. (A) Mass spectrometry analysis. The LANA protein complex was isolated from HeLa nuclear extracts. 3×Flag-tagged LANA was stably expressed in HeLa cells, and nuclear extracts were prepared from 8 liters of suspension culture. Stably Flag-expressing control cells were similarly prepared for a background control. Protein complexes were subjected to SDS-PAGE on a 4-to-16% gradient gel, and the gel was stained with Sypro ruby. Some of the interacting partners that were identified by at least three unique peptides and a total of more than 10 reads with a confidence interval equal to 100% (Mascot/Scaffold program) are listed. hnRNPK, heterogeneous nuclear ribonucleoprotein K. (B) Coimmunoprecipitation of KDM3A and LANA in 293T cells (a) or BCBL-1 cells (b). HEK 293T cells were transiently cotransfected with HA-KDM3A and the Flag control or Flag-LANA. Cell lysates (500 μg) were immunoprecipitated with Flag agarose beads and immunoblotted with anti-HA (a). BCBL-1 cell lysates (1 mg) were immunoprecipitated with rabbit IgG control or anti-KDM3A rabbit IgG and immunoblotted with anti-LANA rat IgG (b). W.B., Western blotting. (C) Colocalization. Immunofluorescence staining of anti-LANA (green) and anti-KDM3A (red) in naturally infected BCBL-1 cells shows colocalization (yellow). Images were taken by using a wide-field 3D deconvolution fluorescence microscope, and 3D image visualization and quantitation were performed by using the VoloCITY digital imaging suite.