Ab2187

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.

For immunoblotting analysis, cells were lysed with NETN (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, 1.25 mM DTT and Roche Protease Inhibitor Mixture) using several freeze/thaw cycles, or sonication (QSonica Q800RS ultrasonic horn). Blots of these extracts were probed with antibodies against the Flag epitope (Sigma A8592), BRCA1 (Abcam, ab16780), RNF168 (Millipore, 06-1130), RNF8 (Santa Cruz Biotech sc-271462), actin (Sigma, A2066), FANCD2 (Abcam, ab2187) and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, sc-2004 and sc-2005). To examine H2AX, H2ax^−/− mouse embryonic stem cells were transfected with expression vectors for RNF168 and/or H2AX using 400 ng each plasmid transfected with 7.2 μl of Lipofectamine 2000 in a 0.4 ml total volume for 3 h. Subsequent to transfection (40 h), cells were treated with 10 Gy IR (Gammacell 3000 irradiator) and allowed to recover 2.5 h prior to lysis with NETN supplemented with 10 mM N-Ethylmaleimide, treated with two freeze/thaw cycles, and centrifuged at 4°C for 10 min. Histones were extracted from the pellet using 0.2 N HCl for 40 min at 4°C for immunoblots that were probed with an anti-H2AX antibody (Millipore 07-627, 1:1000), and developed with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology sc-2004, 1:1000). HRP signals were visualized using ECL reagent (Amersham Biosciences).

Cells grown on glass cover slips were fixed in 4% formaldehyde supplemented with 0.1% Triton X100 for 10 min at room temperature before permeabilization in 0.5% Triton X100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies against FANCD2 (ab2187, Abcam), PhosphoDNA-PKcs (Ser2056 ab18192, Abcam), MDC1 (ab11169, Abcam), γH2AX (JBW301, Upstate), 53BP1 (MAB3802, Millipore), Mre11 (GTX70212, GeneTex), Rad51 (PC130, Calbiochem), RIF1 (A300–569A, Bethyl), RAP80 (NBP1–87156, Novus) and acetylated lysine (MA1–2021, Thermo). Primary antibody detection was achieved by incubation with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 secondary antibodies (Invitrogen) for 30 min at room temperature. Slides were mounted in DAKO mounting medium supplemented with DAPI (Sigma) and examined at a magnification of 63x using a fluorescence microscope (Zeiss Axio Observer Z1) equipped with an ORCA-ER camera (Hamamatsu). The microscope and camera parameters were set for each series of experiments to avoid signal saturation. Image processing and analysis were performed using ImageJ software (http://rsb.info.nih.gov/ij/).