D4w1u

Cells were lysed with 2% SDS in PBS. Protein concentrations were determined with the DC protein assay (BioRad). Equal amounts of proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting with antibodies against 53BP1 (AbCam, catalog number Ab36823; 1 µg/mL), histone H2A (Cell Signaling, clone D603A; 1:1,000), histone H2B (AbCam, catalog number Ab1790; 1:1,000), histone H3 (AbCam, clone 1B1B2; 1:500), H3K9me3 (Cell Signaling, clone D4W1U; 1:800), KU80 (Abcam, catalog number ab119935; 1 µg/mL), laminB (AbCam, catalog number Ab16048; 0.2 µg/mL), and Rad51 (AbCam, catalog number Ab63801; 1:1,000). Enhanced chemiluminescence signals were detected with an Amersham Imager 600.

Samples were rinsed with PBS, fixed 20 min with 10% formalin, washed with PBS glycine (50 mM), permeabilized 10 min with 0.5% Triton X-100, and blocked with 10% goat serum in immunofluorescence (IF) buffer (130 mM NaCl, 13.2 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 0.1% bovine serum albumin, 0.05% NaN₃, 0.2% Triton X-100, and 0.05% Tween 20). Samples were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. After three washes with IF buffer, samples were incubated with secondary antibodies (4 µg/mL) and washed again with IF. Primary antibodies were against γH2AX (Millipore, clone JBW301; 2 µg/mL), H3K9me3 (Cell Signaling, clone D4W1U; 1:800), KU80 (Abcam, catalog number ab119935; 2 µg/mL), laminA/C (Santa Cruz, catalog number SC-7292; 4 µg/mL), NuMA (clone B1C11, a gift from J. Nickerson, University of Massachusetts; 1:2 dilution), and Rad51 (AbCam, catalog number Ab63801; 1:300). Secondary antibodies were anti-mouse AlexaFluor-488 and anti-rabbit AlexaFluor-568 (both from Life Technologies; 1:500). Cell labeling with 5-ethynyl-2′-deoxyuridine was done using Click-iT reagents (Invitrogen). Nuclei were stained with 0.5 µg/mL DAPI. Samples were mounted using ProLong Gold antifade (Molecular Probes) and imaged with an IX83 Olympus microscope, using a 60× oil immersion objective (NA = 1.35) and the appropriate filter cubes (Chroma).