Blocker bsa bovine serum albumin buffer

Cells were seeded onto coverslips and cultured overnight. The adhered cells were washed twice with cold PBS for 5 min and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then washed three times with PBS for 5 min each and permeabilized with ice-cold 0.5% NP-40 in PBS for 10 min on ice. Cells were washed three times with PBS for 5 min each and incubated with anti-phosphorylated histone γH2AX antibody (Millipore), followed by Alexa 488 secondary antibody (Molecular Probes) with 30% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific). After staining, labeled protein was cross-linked with 4% paraformaldehyde in PBS for 20 min at room temperature. The samples were washed two times with PBS for 5 min and then dehydrated in 70, 90, and 100% ethanol for 3 min each and air-dried. Hybridization mixtures (10 μl) containing 3 ng of fluorescence-labeled telomeric PNA probe were applied to the slide and mounted under a coverslip. The slides were heated for 3 min on a hot plate at 80 °C. After hybridization with a telomeric PNA probe for 5 h, the cells were washed three times with 70% formamide/10 mM Tris (pH 6.8) for 15 min, followed by a 5-min wash with 0.05 M Tris/0.15 M NaCl (pH 7.5)/0.05% Tween-20 and a 5-min wash with PBS. Mounting and microscopic analysis was performed as for the telomere FISH analysis.

Cell lysates were prepared in Laemmli buffer containing benzonase nuclease (Sigma), complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and size-fractionated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted to Immobilon-P nylon membrane (Millipore). Membranes were blocked with 10% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific) and incubated with the primary antibodies overnight at 4 °C. After incubation with each appropriate secondary antibody, bands were detected with ECL (GE Healthcare) using X-ray films. Antibodies were diluted in 10% Blocker BSA buffer (Thermo Fisher Scientific). Antibodies used in this study are listed in Supplementary Data 4.