Anti p atr

The inhibitors used in this study were as follows: 100 nM UCN01 and 5 µM VE822 (Selleckchem, Houston, TX); 10 µM pimozide (Sigma-Aldrich, St. Louis, MO); and 5 µM CHK2i (Calbiochem, San Diego, CA). The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA). For Western blot analysis, cell lysates were processed as previously described (36 (link)). Briefly, keratinocytes were first isolated from J2 feeders by treatment with Versene (phosphate-buffered saline [PBS] containing 0.5 mM EDTA) and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min. The protein samples were separated on SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were finally developed using Enhanced Chemiluminescence (ECL) Prime or ECL reagents (Amersham, Pittsburgh, PA). Chemiluminescence signals were detected using Eastman Kodak X-ray films.

HFKs as well as CIN612 HPV31-positive keratinocytes were differentiated in raft cultures as previously described (35 (link)). Sections from HFK raft cultures or CIN612 cell raft cultures were processed by the Pathology Core Facility of Northwestern University. Cross sections of rafts were stained using a 1:100 dilution of anti-p-ATR (Cell Signaling, Inc., Danvers, MA) and 1:100 of anti-CHK1 (Cell Signaling, Inc., Danvers, MA). For the secondary antibody, Alexa Fluor 488 goat anti-mouse antibody and anti-rabbit antibody were used (Life Technologies, Grand Island, NY). The sections were then mounted with Gelvatol (Sigma-Aldrich, St. Louis, MO). Images were captured using a Nikon C2+ spectral confocal microscope at Center of Advanced Microscopy of Northwestern University.