Fig 1.

HSP90 interacts with HTLV-1 Tax. (A) 293T cells were transfected with pNTAP-Tax, pRL-tk, and NF-κB-luciferase plasmids. After 24 h, cells were lysed and subjected to a dual-luciferase assay. Lysates were subjected to immunoblotting with anti-Tax and antiactin. (B) 293T cells were transiently transfected with pNTAP-Tax or pNTAP empty vector. Tandem affinity purification was performed using sequential purification steps with streptavidin and calmodulin resin. The purified Tax and associated protein complexes were resolved by SDS-PAGE and silver staining. The bands indicated by the arrows were excised and subjected to LC-MS/MS analysis. (C) 293T cells were transfected with Flag-Tax and HA-HSP90 plasmids as indicated. After 24 h, cell lysates were immunoprecipitated with Flag antibody and immunoblotting was performed with anti-HA and anti-Flag (top panel). The reciprocal IP was also performed with HA antibody and immunoblotting with anti-Flag (bottom panel). Lysates were subjected to immunoblotting with anti-HA, anti-Flag, and antiactin. (D) MT-2 cells were treated with the HSP90 inhibitor 17-DMAG (0.5 μM) for 2 h as indicated. Cell lysates were immunoprecipitated with HSP90 beta antibody, and immunoblotting was performed with anti-Tax and anti-HSP90 beta. (E) Jurkat, MT-2, C8166, and MT-4 cells were lysed and subjected to immunoblotting with anti-HSP90, anti-Tax, and antiactin. (F) 293T cells were transfected with GFP-Tax and HA-HSP90 plasmids. After 24 h, cells were stained with DAPI, phalloidin, and anti-HA and subjected to confocal microscopy. (G) C8166 cells were stained with DAPI, anti-Tax, and anti-HSP90 and subjected to confocal microscopy.