Skip to main content
. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: J Virol Methods. 2017 Jun 8;247:99–106. doi: 10.1016/j.jviromet.2017.04.019

Fig. 6. Ectopic Rta, but not NICD1, is sufficient to reactivate KSHV from latency.

Fig. 6

(A). Cells were transfected with plasmids expressing genomic Rta (Rta) (2.5ug), mutant Rta (RtaΔSTAD)(2.0 ug), or Notch intracellular domain isoform 1 (NICD1)(0.5 ug). 72 h. post transfection, viral media was harvested and transferred to 293 reporter cells, which were returned to the incubator for 72 h. prior to assaying the indicated volumes of media for alkaline phosphatase using fluorescence. Alkaline phosphatase values calculated from media were subtracted from sample values and all samples were normalized to empty vector alone, which was set at 1 (B), (C), (D) Western blots from extracts of Vero rKSHV.294 cells transfected with each of the plasmids in part A; plasmid expressing histone deacetylase 1 (HDAC1) was transfected as a positive control for the FLAG antibody in the western blot. Total cellular proteins were extracted 48 h post-transfection using RIPA buffer and resolved on a 10% SDS polyacrylamide gel. Primary antibodies were specific for FLAG-M2 (Sigma, F1804-200UG) and tubulin (Sigma, T6199) (B), Rta and GAPDH (C), and V5 and GAPDH (D). The arrow in (C) indicates the Rta band; the lower band in (C) is a background band consistently detected when using the Rta-specific antibody. Numbers to the left of blots indicate migration positions of molecular weight standards; kDa= kilodaltons. (*p < 0.05 as determined by multifactorial Anova and Tukey post-hoc).