mm post ns mm post ns mm

35 everolimus 711–1254/29/084 mm-2 post, 300 ns36
35971354/29/084 mm-2 post, 300 ns36
3752149/24/0810/08/0810/22/084 mm-2 post, 300 ns43
503317.41/14/091/20/091/28/05Needle, 100 ns44
5033241/14/091/20/092/3/096-postsing, 100 ns44
5033542/3/092/16/096-postsing, 100 ns, 10 hz24
5033642/3/092/16/096-postdual, 100 ns, 7 hz24
5033752/16/096-postdual, 100 ns, 7 hz11
5033852/16/096-postsing, 100 ns, 7 hz11
504014.51/14/096-post sing, 100 ns6
5040231/20/091/28/09Needle, 1776p; 769 p16
5040342/3/096-postdual, 100 ns2
50404101/28/09Needle, 2705 p, 100 ns8
5041261/26/092/13/096-postdual, 100 nsPoor fix
5041451/26/092/13/096-postsing, 100 nsPoor fix
5042241/26/092/13/096-postdual, 100 ns39
5042441/26/092/13/096-postsing, 100 ns39
5034162/3/092/16/096-postsing, 100 ns24
5034382/3/092/20/098 mm needle, 100 ns24
5034452/20/094 mm needle, 100 ns7
5034542/20/094 mmneedle, 100 ns7
575543.512/8/096-post dual, 100 ns15
575613.51/6/106-post dual, 100 zone of intolerance ns27
5756241/6/106-post dual, 100 ns27
5756331/11/106-post dual, 100 ns13

Chromatin immunoprecipitation ChIP was performed

Chromatin immunoprecipitation (ChIP) was performed essentially as described previously [28]. Briefly, GW2580 were harvested and incubated in 1% formaldehyde for 15 min to crosslink proteins to DNA, and the reaction was quenched by incubating cells in 125 mM glycine for 5 min. Cells were lysed with glass beads, and extracts were sonicated to shear DNA to an average size of 0.5 kb. Extracts were divided into two aliquots of input DNA and immunoprecipitation (IP) DNA, at a ratio of 1:20. Immunoprecipitation was carried out with a monoclonal anti-Myc antibody (9E10) (Santa Cruz Biotechnology, Inc.), and immune complexes were captured by Dynabeads Protein G (Dynal Biotech) for 4 h at 4 °C. After a series of washes, proteins were released from the beads by incubation for 6 h at 65 °C and treated with Proteinase K. The DNA was purified, and precipitated DNA was quantified with real-time PCR with SYBR green (TaKaRa Thermal Cycle Dice Real Time System). The sequence information of the primers used is available upon request.

Antiviral activity Vero cells grown in well

2.5. Antiviral activity
Vero cells grown in 96-well tissue culture plates were infected with HSV-1 (strains KOS or B2006) or VSV at moi of 0.07 PFU/cell. After 1 h adsorption at 37 °C the inoculum was removed and medium containing different concentrations of compounds 4a–j was added, by triplicate. The plates were incubated at 37 °C in a 5% CO2 Adriamycin until 100% cell death was observed microscopically in untreated infected control cells. Supernatants corresponding to those triplicates were harvested after cell disruption by three cycles of freezing and thawing and pooled. Virus yields were titrated by plaque assay. For comparative purposes, acyclovir (ACV) was tested as positive control against HSV-1.
2.6. Indirect immunofluorescence assay
For total glycoprotein staining, subconfluent cells grown on glass coverslips in 24-well plates were fixed with methanol for 10 min at ?20 °C. After washes with phosphate buffered saline (PBS), the coverslips were incubated with primary antibody for 30 min at 37 °C, and then returned to culture dishes and subjected to additional washes with PBS. Afterwards, cells were incubated with secondary antibody for 30 min at 37 °C. Finally, coverslips were rinsed, mounted and photographed with an Olympus BX51 microscope with epifluorescence optics. The mouse monoclonal antibody anti-gD of HSV-1 was obtained from Santa Cruz Biotechnology, USA. The rabbit polyclonal anti-gG of VSV was kindly provided by Dr. Pablo Grigera (CEVAN Buenos Aires, Argentina). Secondary goat anti-rabbit FluoroLinkTM CyTM2 and antimouse FluoroLinkTM CyTM3 antibodies were purchased from GE Healthcare Bio-Sciences, Argentina.