KSHV vGPCR causes decreased levels of cyclin A, D2 complexes, and decreased cdk2 kinase activity. (A) To express vGPCR, BC3.14 cells were exposed to 2 μg/mL doxycycline for the time shown in hours. Protein lysates were run on 10% SDS-PAGE gels and probed with appropriate antibodies. (B) BC3.14 cells were incubated for 48 hours with doxycycline and then lysed in kinase buffer. Antibodies were then used to immunoprecipitate each kinase as indicated. After resuspension in kinase buffer, the substrate GST-pRb (C term) was added along with [γ-³²P] ATP. Phosphorylated product was detected by autoradiography after separation on a 12% SDS-PAGE gel (top). Ten percent of the protein input for the IP was used to Western blot for each kinase (bottom). A representative of 3 independent experiments is shown.