α γh2ax

To detect FANCD2, whole cell extracts prepared as described previously [47 (link), 48 (link)] were resolved in pre-cast Tris-acetate gels (Life Technologies), transferred onto nitrocellulose membranes and probed with the α-FANCD2 antibody ab2187 (Abcam). Mouse α-CHK1 antibody was from Santa Cruz (Cat. No. sc-8408). Phosphorylation of CHK1 and CHK2 was analyzed with a Phospho-Chk1/2 Antibody Sampler Kit (Cell Signaling, Cat. No. 9931). Other antibodies were: α-PCNA Cat. No. sc-56 (Santa Cruz), α-RPA32 Cat. No. A300-244A (Bethyl Laboratories), α-γH2AX Cat. No. 05-636 (Millipore), α-Nucleolin Cat. No. 396400 (Life technologies). Biotin-conjugated EdU was visualized in dot blots with HRP-conjugated α-biotin antibody, Cat. No. 7075 (Cell Signaling). All proteins were visualized by ECL (Amersham or Thermo Scientific) and quantified using Storm Phosphoimager and ImageQuant software (Molecular Dynamics) or FluorChem Imager (Alpha Innotech), using manufacturer-supplied software. For presentation, images were saved in TIFF format, adjusted for brightness/contrast and cropped using Adobe Photoshop, and assembled into figures in CorelDraw. Brightness/contrast adjustments were made to entire images. In some cases an image of one and the same blot was cut and spliced to delete extraneous lanes or to change the order of lanes.