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. 2015 Nov 20;11(11):e1005274. doi: 10.1371/journal.ppat.1005274

Fig 1. Successful enrichment of the nuclear envelope region and associated KSHV RTCs in HEK-293T rKSHV.219 cells.

Fig 1

Successful enrichment of NE regions in unreactivated (-) and reactivated for 48 h (+) HEK-293T rKSHV.219 cells was demonstrated by Western blotting. Equal amounts of total protein extracted from whole cell (WC) and nuclear envelope (NE) fractions were used. The monoclonal antibody Mab414 specifically recognises the phenylalanine-glycine (FG)-repeat motif present in numerous nucleoporins (Nups). Closed arrows point to Nups that were only detected by MAb414 after NE enrichment. Open arrow points to a nucleoporin lost during NE enrichment. Several FG Nups, together with Nup160, histone H3 and lamin B1 were all markedly enriched in the NE fractions compared with WC fractions. The nucleolar proteins B-23 and C-23 and the cytoplasmic GAPDH protein were all decreased in NEs fractions indicating correct subcellular fractionation. The viral RTC-associated ORF57 and RTA proteins were used to monitor lytic reactivation and assess RTC enrichment. ORF57 antibody detected both full length ORF57 protein (FL-ORF57) and the caspase-7-cleaved ORF57 (CL-ORF57).