FIG 2.
K1 is required for Akt phosphorylation and lytic gene expression in reactivated cells. (A and B) A total of 5 × 105 WT KSHV-, KSHV-K1FLAG-, KSHV-K15×STOP-, KSHV-K1REV-, and KSHVΔK1-infected iSLK cells were seeded overnight and then treated with 1.5 μg/ml of doxycycline for 72 h (A) or for 24, 48, and 72 h (B). Cells were harvested, lysed, and subjected to Western blot analysis with an anti-FLAG antibody to detect K1, an anti-pAkt (S473) antibody to detect phosphorylated Akt, an anti-vIL-6 antibody to detect vIL-6, an anti-K8α antibody to detect K8α, and an anti-ORF45 antibody to detect ORF45. (C) A total of 2 × 105 cells were seeded overnight and then treated with 3 μg/ml of doxycycline for 72 h. Cells were fixed with 3.7% formaldehyde and subjected to an immunofluorescence assay with primary anti-ORF59 antibody (1:500; Advanced Biotechnologies) and secondary anti-mouse IgG antibody conjugated to tetramethyl rhodamine isocyanate (1:500). (D) Graph depicting the number of quantitated ORF59-positive cells per field following reactivation.