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. 2015 Mar 4;89(10):5536–5556. doi: 10.1128/JVI.03713-14

FIG 1.

FIG 1

FIG 1

LANA interacts and colocalizes with the components of the RFX complex. (A) Coimmunoprecipitation of LANA with the components of the RFX complex, RFX5, RFXAP, and RFXANK, in KSHV-positive BCBL-1 and BC-3 cells. LANA was immunoprecipitated (IP) by using mouse anti-LANA antibody from precleared cellular lysates of nearly 20 million BCBL-1 or BC-3 cells. The coimmunoprecipitated proteins were analyzed by SDS-PAGE followed by immunodetection using antibodies specific for RFX5, RFXAP, or RFXANK. An isogenic antibody, mouse IgG (CoAb), was used as a control for immunoprecipitation in this assay. IB, immunoblotting. (B) Colocalization of LANA with individual components of the RFX complex, RFX5, RFXAP, and RFXANK. LANA was localized by using rat anti-LANA antibody, and the RFX proteins were detected with specific antibodies in BCBL-1, BC-3, and BJAB cells. Secondary antibodies for LANA and the RFX complex were Alexa Fluor 594 (red) and Alexa Fluor 488 (green), respectively. Nuclei were stained with To-Pro3 (blue). LANA colocalized with RFX5, RFXAP, and RFXANK in the nuclei of BCBL-1 and BC-3 cells as punctate dots. Staining of RFX5, RFXAP, and RFXANK in KSHV-negative BJAB cells showed distinct staining. DIC images were captured to show cell morphology. (C) Triple-immunofluorescence staining showing colocalization of RFXAP and RFX5 with LANA in BCBL-1, BC-3, and BJAB cells. LANA was stained with rat anti-LANA, RFXAP was stained with rabbit anti-RFXAP, and RFX5 was stained with mouse anti-RFX5. Host-matched secondary antibodies were used for their localization. The merged panels show the colocalization of all three proteins at certain foci in KSHV-positive cells.