Anti topbp1

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.