The AR interacts with the KSHV receptor EphA2 during the early infection stage.

<p>(a) The AR co-immunoprecipitates with the KSHV entry receptor EphA2 during KSHV infection. SLK cells were serum-starved for 6 h prior to KSHV infection. At the indicated times, uninfected or KSHV-infected cells were immunoprecipitated by an α-AR antibody and analyzed by western blotting. (b, c) The interaction between AR with wild-type EphA2 and AR with EphA2 Ser897Asn mutant were confirmed by the absence of KSHV infection. Plasmids expressing FLAG-tagged AR, wild-type EphA2 or EphA2 Ser897Asn mutant were transfected into 293T cells individually or in indicated combination.48 h later, the cells were subjected to immunoprecipitation using anti-FLAG M2 affinity beads, followed by western blotting. (d) The AR co-localizes with EphA2 in HUVECs. Cells were reacted with α-AR (red) and EphA2 (green) antibodies. Scale bars represent 40 μm. Representative images are shown. (e, f) The AR associates with the intracellular kinase domain of EphA2. Three GST-fused truncations of EphA2 were constructed: EC-EphA2 (the extracellular domain), EC-TM EphA2 (the extracellular domain plus transmembrane domain), and KD-EphA2 (the kinase domain) (e). GST affinity beads were pre-loaded with the GST-fused proteins or GST, and then incubated with in vitro-translated AR. Each reaction was repeated in, at least, triplicate.