Analysis of interaction between LANA and Hsp90.
(A) Interaction between Hsp90 and LANA or respective LANA mutants in HeLa cells. HA-tagged Hsp90 was co-transfected with Flag-tagged full-length LANA (aa1–1162) or indicated mutants: Flag-tagged LANA-N (aa1–329, pDD1928), Flag-tagged LANA-C(aa930–1162,pDD1931), or Flag-tagged LANA N+C (aa1–329 and 928–1162, pDD775). Protein extracts were immunoprecipitated with anti-Flag antibody followed by immunoblotting with anti-HA antibodies, IgG was used as control. Input samples were from cell lysate supernatants. MW markers in kD are indicated on the left. (B) Reverse immunoprecipitation. HA-tagged Hsp90 together with Flag-tagged LANA were co-transfected into HeLa cells, empty pcDNA vector was used for control. Protein extracts were immunoprecipitated with anti-HA antibody and immunoblotted with anti-LANA antibody. Input samples were from cell lysate supernatant. (C) Co-localization analysis of LANA and Hsp90. Immunofluorescence assay was performed after fixation and permeabilization of TIVE-L1 cells, incubated with primary rabbit anti-LANA and mouse anti-Hsp90 antibodies and the secondary anti-rabbit Texas (red) and anti-mouse FITC (green) conjugated antibodies respectively. Nuclear fractions were stained with DAPI, images were observed under fluorescence microscope.