Nuclei ez prep isolation kit

HMVEC-d were starved for 2 h with or without C-646 and either left uninfected or infected with KSHV (30 DNA copies/cell) at 37°C for 2 h. These cells were washed, treated with trypsin-EDTA to remove non-internalized virus, and incubated for varying times of infection [18 (link)]. Nuclei were isolated using a Nuclei EZ Prep isolation kit (Sigma) according to the manufacturer’s instructions. Briefly, cells were lysed on ice for 5 min with a mild lysis buffer (Sigma), and nuclei were concentrated by centrifugation at 500xg for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures as described previously [11 (link)]. DNA was extracted from isolated nuclei using a DNeasy kit (Qiagen, Germantown, MD). Internalized nuclear KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR [2 (link)]. The KSHV ORF73 gene cloned in the pGEM-T vector (Promega) was used for the external standard. The CT values were used to generate the standard curve and to calculate the relative copy numbers of viral DNA in the samples.