Fancd2 nb100 182

Whole cell lysates were prepared using RIPA buffer, separated on 3–8% gradient gels using the NuPage system (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and transferred onto nitrocellulose membrane. Blots were incubated with 1:1000 dilution of the antibodies overnight at 4 °C and developed using enhanced chemiluminescence (Super ECL or West Femto, Thermo Fisher Scientific). The following antibodies were used: FEN1 (sc-13051, Santa Cruz), FANCA (sc-28215, Santa Cruz, Dallas, TX, USA), FANCD2 (NB100-182, Novus, Wrentham, MA, USA), FANCI (A301-254A, Bethyl Labs, Montgomery, TX, USA), β-Tubulin (cs2128, Cell Signaling, Danvers, MA, USA), Vinculin (cs13901, Cell Signaling), goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Santa Cruz Biotech Inc.). Full images of blots are available in the Supplemental Materials.

Antibody to Phospho‐S345‐Chk1 (#2348) and Phospho‐S10‐Histone H3 (#53348) was purchased from Cell Signaling Technology (Danvers, MA, USA); BRCA1 (sc‐6954), Cyclin A (sc‐2712682), Chk1 (sc‐8408), and ATR (sc‐1887) from Santa Cruz (Dallas, TX, USA); p53 (Ab‐6, Clone DO‐1, MS‐187‐P0) from Thermo Fisher Scientific; 53BP1 (MAB3802) and γH2AX S139 (05‐636) from MilliporeSigma (Burlington, MA, USA); and FANCD2 (NB100‐182) from Novus Biologicals (Centennial, CO, USA). The polyclonal anti‐Timeless antibody was generated previously [28 (link)].