Sc 23921

Appropriate amounts of proteins (30 μg or 60 μg), extracted from a pool of 3 P1-cerebella and MEFs were loaded and separated by SDS-PAGE. Proteins were electro transferred to PVDF or nitrocellulose membranes (Trans-Blot Turbo Transfer Pack, BIO-RAD Laboratories, Hercules, CA) at the Trans-Blot Turbo Transfer System (BIO-RAD Laboratories). After blocking, membranes were incubated over night at +4°C with primary antibodies against DNA-PKcs and p53 (ab70250 and ab31333, Abcam, Cambridge, UK), ATM, p21 (sc-23921 and sc-471, Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(Ser18) (9284, Cell Signaling Technology, Inc., Danvers, MA), β-actin and HSP70 (A5316 and H5147, Sigma-Aldrich, St Louis, MO). Membranes were probed for 1h at RT with appropriated HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were visualized using Amersham ECL Prime WB detection reagent (GE Healthcare Europe, Milan, Italy). Images were acquired using a Image 6 quant LAS 500 (GE Healthcare Europe), and densitometric analysis was performed using ImageJ software.

SW620, DLD1, HCT-116 and HCT-116 chr3 cells were trypsinised, collected and lysed in Cell Panel Lysis Buffer with complete protease and phosphotase inhibitors. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membrane by Western blot. Membranes were probed, at a concentration of 1:1000 unless stated otherwise, for ATM (sc-23921, Santa Cruz Biotechnology), MSH2 (556349, BD Pharmigen), MLH1 (WH0004292M2, Sigma Aldrich), MSH6 (610918, BD Transduction), MRE11A (ab214, Abcam), RAD50 (611010, BD Transduction), NBS1 (NB100-143, Novus Biologicals), FEN1 (ab109132, Abcam) and β-Actin (A5441, Sigma Aldrich).