Fig. 4

Visualization of LANA dots and Western blot analysis of LANA 16 days following targeting of orf73 by CRISPR-Cas9. Cas9-BAC16-mCherryORF45-infected SLK cells were transduced with recombinant lentiviruses expressing a random non-targeting sgRNA or orf73 sgRNAs. Cells were maintained with no hygromycin selection, fixed 16 days post transduction and stained with rat monoclonal anti-LANA followed by anti-rat Alexa Fluor 647-conjugated secondary antibodies. The corresponding staining of nuclear DNA by Hoechst is also displayed (a). BAC16-mCherryORF45-infected SLK cells expressing Cas9 were treated as described in Fig. 1. Cells were collected 16 days post transduction, and protein extracts were prepared. Samples containing 60 µg of protein extracts from cells that were maintained with hygromycin and with no hygromycin selection, respectively, were used to determine LANA protein expression while Tubulin was used as loading control (b)