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. 2009 Nov 11;38(2):467–476. doi: 10.1093/nar/gkp929

Figure 5.

Figure 5.

Short telomeric substrates activate a DSB response and are repaired by NHEJ. (A) Southern blot analysis of NHEJ-mediated repair products. In total, 0.6 kb of untagged NT or telomeric DNA was added to cytosolic extracts for 150 min. Reactions were processed for native agarose gel electrophoresis before transfer onto a Nitrocellulose membrane. Telomeric substrates are hybridized with a 5′ 32P-labeled (CCCTAA)6 oligonucleotide and NT substrates with random labeled pRST5_NT. LM linear monomer, LD linear dimer, M multimers. Signals from three independent experiments were quantified by phosphorimager analysis and expressed as ratios of the LD to LM. (B) Immobilized NT or telomeric DNA was incubated with cytosolic extracts at indicated concentrations and activation of DNA damage response proteins in the soluble fraction was monitored by western blotting. Lane 9, buffer control, lane 10, 2 ng/µl HaeII-digested pBS was added. (C) Immobilized NT or telomeric DNA was incubated with cytosolic extracts at 2 × 109 DSB/µl and checkpoint activation in the soluble fraction was monitored by western blotting using an antibody against phosphorylated Chk1. Beads only, lane 5, 2 ng/µl HaeII digested pBS, lane 6, buffer control, lane 7.