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. 2009 Jul 13;29(18):4918–4934. doi: 10.1128/MCB.00009-09

FIG. 2.

FIG. 2.

(A) Overexpresssion of TRF2-RK induces TIF formation. Indirect immunofluorescence using anti-TRF1 in conjunction with anti-53BP1 was performed with fixed hTERT-BJ cells expressing either TRF2-RK or the vector alone. Arrowheads indicate sites of colocalization of 53BP1 and TRF1. (B) Quantification of percentage of cells with more than five TIFs. For each cell line, a total of more than 800 cells from three independent experiments were scored. Standard deviations derived from three independent experiments are indicated. (C) Dot blots of ChIPs. ChIPs were performed with either anti-TIN2 or anti-IgG antibody in cell extracts from HT1080 cells overexpressing TRF2-RK or the vector alone. Precipitated DNA was analyzed for the presence of TTAGGG repeats and Alu repeats by Southern blotting. (D) Quantification of anti-TIN2 ChIPs. The signals were quantified by ImageQuant analysis. The percentage of precipitated DNA was calculated relative to the input signals. Standard deviations derived from three independent experiments are indicated. (E) Western blot analysis of TRF2 protein expression. Whole-cell extracts made from 200,000 cells were used, and immunoblotting was performed with anti-TRF2 and anti-TIN2 antibodies. The γ-tubulin blot was used as a loading control. (F) Genomic blot of telomeric restriction fragments of hTERT-BJ cells expressing various proteins as indicated above the blot. About 3 μg of RsaI/HinfI-digested genomic DNA from each sample was used for gel electrophoresis. The DNA molecular size markers are shown on the left of the blot. The bottom panel, taken from an ethidium bromide-stained agarose gel, is used as a loading control. (G) Genomic blot of telomeric restriction fragments of hTERT-BJ cells expressing various proteins as indicated above the blot. About 3 μg of RsaI/HinfI-digested genomic DNA from each sample was separated on a CHEF gel. The picture of an ethidium bromide (EtBr)-staining gel is shown at the right of the blot, whereas the DNA molecular size markers are shown at the left of the blot.