Anti rpa2 ps4 8

Cell lysates were prepared in RIPA buffer, (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 1% (v/v) NP40, 0.1% (w/v) SDS and 0.5% (w/v) sodium deoxycholate, supplemented with protease inhibitors tablets and Phostop tablets) for 30 minutes on ice. Resulting lysates were clarified at 16,000 × g for 20 minutes. Gel electrophoresis was performed using the NuPAGE system (Invitrogen). Briefly, samples were resolved on 4–12% Bis-Tris gels in MOPS buffer, transferred to a PVDF membrane. Membranes were incubated with antibodies diluted in PBS supplemented with 5% milk protein and incubated, with agitation, overnight at 4° C. Membranes were washed in PBS for 3 × 5 minutes and incubated in appropriate secondary antibodies for 1 hour at room temperature. The antibodies used for immunoblotting were as follows: anti-CDK18 (Santa Cruz Biotechnology: sc-176), anti-pCDK substrate ([K/H]pSP) MultiMab rabbit Ab mix (Cell Signalling), anti-CHK1 (Sigma Aldrich), anti-CHK1 pS317 (Cell Signaling), anti-RAD9 (Bethyl laboratories: A300–800A), anti-RPA2 (Calbiochem), anti-RPA2 pT21 (Abcam), anti-RPA2 pS4/8 (Bethyl Laboratories), anti-KAP1 (Bethyl Laboratories), anti-KAP1 pS824 (Bethyl Laboratories), anti-ATR (Santa Cruz Biotechnology), anti-Actin (Abcam), HRP-secondary antibodies (DAKO) and Alexa-Fluor antibodies (Invitrogen).