FIG. 1.

In vivo interaction of K8 and CBP. (A) The coimmunoprecipitation of in vivo-synthesized K8 and CBP was analyzed by Western blotting. 293T cells were transfected with an HA-tagged CBP expression vector and either a blank (pEBG) vector or an expression vector carrying GST-fused K8 (pEBG-K8). The cells were harvested, lysed, and precipitated with either anti-HA antibody (α-HA) or anti-GST antibody (α-GST), and protein G resin. The proteins were analyzed by SDS-PAGE and immunoblotted with anti-HA or anti-GST antibodies. (B) Coimmunoprecipitation assay in the KSHV-positive BCBL-1 cell lines. The BJAB cell lines were used as a KSHV-negative control. BCBL-1 cell lines, with or without TPA induction (48 h), were harvested, and the appropriate lysates were precipitated and immunoblotted with either CBP-specific monoclonal antibody (α-CBP) or rabbit polyclonal anti-K8 antibody (α-K8), respectively. (C) K8 colocalizes with CBP in 293T cells. The GFP-K8 and HA-CBP expression vectors were transfected into 293T cells. Cells were fixed and immunostained 48 h after transfection. HA-CBP was detected using a rhodamine-conjugated secondary antibody against a mouse monoclonal HA antibody. (D) Colocalization of K8 and CBP in BCBL-1 cells. For the expression of lytic gene product K8, BCBL-1 cells were treated with TPA for 48 h. K8 was detected by indirect immunofluorescence using a rabbit anti-K8 antibody as a primary antibody and FITC-conjugated goat anti-rabbit IgG as a secondary antibody. CBP was detected with a mouse CBP-specific monoclonal antibody and TRITC-conjugated goat anti-mouse IgG. The nucleus of the cell was stained with DAPI.