Lack of CTCF or Rad21 effect on ORF50/RTA levels or ORF50/RTA activation function.

<p>A. KSHV-infected iSLK cells were induced to replicate after control KD (NC Si), CTCF KD (CTCF Si) or Rad21 KD (Rad21 Si). Cells were either induced to permit KSHV replication with doxycycline (+D) or mock-induced (−D). Protein lysates were prepared at 24 hours post-induction and blotted with anti-ORF50/RTA antibodies. B. Samples were prepared from cells treated as in (A) above but harvested at 48 h post-induction and analyzed by immunoblotting with ORF50/RTA antibodies. C. Effect of CTCF or Rad21 depletion on ORF50/RTA transcriptional activation function. 293 cells were depleted of CTCF (CTCF Si) or Rad21 (Rad21 Si) by siRNA transfection or transfected with a negative control siRNA (NC Si). 48 h after siRNA transfection, cells were transfected with an ORF57 promoter-luciferase reporter plasmid (DD398) and either ORF50 expression plasmid (ORF50) or empty vector (vector). Luciferase assays were performed 48 h after transfection and are expressed as relative luminescence units. D. Lysates of cells used for luciferase assays in (C) above were immunoblotted with anti-CTCF or anti-Rad21 antibodies to confirm complete depletion.