Yy1 sc 1703

Western blotting was performed as previously described [23] (link). Commercially-obtained antibodies against the following proteins were used: (i) ATR (sc-1887), ATM (sc-23921), Chk1 (sc-56291), p-Chk1/Ser345 (sc-17922R), DNA-PKcs (sc-1552), and YY1 (sc-1703) from Santa Cruz (Santa Cruz, CA); (ii) ATRIP (2737) from Cell Signaling Technology (Danvers, MA); (iii) actin (ab8227-50) from Abcam (Toronto, Canada); (iv) GAPDH (MAB374) and γH2AX/Ser139 (JBW301) from Millipore (Billerica, MA).

Briefly, nuclear and cytoplasm extracts were prepared using a nuclear extraction kit (Bio-Rad, Hercules, CA, USA). The samples were loaded onto 10% SDS polyacrylamide gels and electrophoresed (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane and incubated with rabbit polyclonal antibodies against StAR (sc-25806, 1:800 dilution) (Santa Cruz, CA, USA), YY1 (sc-1703, 1:800 dilution) (Santa Cruz, CA, USA), GAPDH (sc-25778, 1:2000 dilution) (Santa Cruz, CA, USA), and TBP (ab63766, 1:2000 dilution) (Abcam, Cambridge, MA, USA) overnight at 4 °C. Finally, the membrane was incubated in the dark for 1 h with a fluorescent secondary antibody and specific bands were detected via electrochemiluminescence (ECL) assay. The signals of StAR (30 kDa), YY1 (68 kDa), GAPDH (37 kDa), and TBP (38 kDa) were visualized using ECL reagents (Pierce Biotechnology, Inc. Rockford, IL, USA) and then captured using a chemiluminescence and multicolor fluorescence imaging system (Fusion Fx7, Vilber Lourmat, Marne la Vallée Cedex 1, France). Specific bands were analyzed using Gel-Pro Analyzer 4.0 (Media Cybernetics, Silver Spring, MD, USA).