Evidence for UL33 and UL78 heteromerization with CCR5 and CXCR4. (A-B) Detection of v7TM/chemokine receptor heteromers using co-IP. Human embryonic kidney (HEK) 293T cells were transfected with HA-UL33-Rluc (A) or HA-UL78-Rluc (B) in presence or absence of CCR5-YFP, CXCR4-YFP, V2-YFP, or Flag-UL33-YFP (A) or Flag-UL78-YFP (B). Lysates were immunoprecipitated with rat anti-HA, resolved by SDS-PAGE and immunoblotted with mouse anti-GFP antibodies (top blot). Expression levels of YFP fusion proteins were assessed by immunoblotting with mouse anti-GFP antibodies (middle blot) and expression of Rluc fusion proteins by monitoring Renilla luciferase activity (bottom graph). (C-D) BRET donor saturation curves were performed by cotransfecting a fixed amount of HA-UL33-Rluc (C) or HA-UL78-Rluc (D) in presence of increasing amounts of CCR5-YFP, CXCR4-YFP, V2-YFP, or Flag-UL33-YFP (C) or Flag-UL78-YFP (D) in HEK 293T cells. The saturation curves are obtained from 3 independent experiments. Vertical lines have been inserted to indicate repositioned gel lanes.