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. Author manuscript; available in PMC: 2011 Aug 1.

Published in final edited form as: Aging Cell. 2010 May 10;9(4):580–591. doi: 10.1111/j.1474-9726.2010.00585.x

Fig. 6 — (A) Dnmt3b is localized in the chromatin fraction of control and WRNp-deficient cells. C_V and WRNp-deficient cells (clone 61) were treated with 10 µM RA for 3 days. Chromatin fraction was then separated and both chromatin-lacking lysates (L) and the chromatin fraction (Ch) were analyzed by western blotting. H3 – histone H3, loading control for the chromatin fraction, Trim5α – cytoplasmic fraction loading control. (B) Dnmt3b is present in WRNp immunoprecipitates. Top: WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin-lacking lysates and from the chromatin fraction of undifferentiated and RA treated cells. WRNp immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), D3 – chromatin input lysate immunoprecipitated with the Dntm3b antibody, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. Bottom: Control cells and WRNp-deficient cells (clone 61) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin fraction of undifferentiated and RA treated cells. (C) WRNp is present in Dnmt3b immunoprecipitates. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp and Dnmt3b then were immunoprecipitated from the chromatin fraction. Immunoprecipitates were resolved on SDS-PAGE and WRNp was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), W – lysates immunoprecipitated with a WRNp antibody, D3 - lysates immunoprecipitated with a Dnmt3b antibody, I – lysates immunoprecipitated with normal rabbit IgG. (D) Effect of EtBr and DNAse on Dnmt3b and WRNp co-immunoprecipitation. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp was immunoprecipitated from the chromatin fraction as in Fig. 6B and C, except cell lysates were treated with EtBr (100 µg/ml) or DNAse prior to immunoprecipitation (see Methods). Immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. Et – EtBr-treated lysates, Ds – DNAse-treated lysates, M – mock-treated lysates, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. (E) Q-ChIP analysis of Dnmt3b levels at the Oct4 promoter regions of control and WRNp-deficient cells. Cells were treated with RA for three days as described above and then subjected to Q-ChIP analysis using a Dnmt3b antibody.

Fig. 6 — (A) Dnmt3b is localized in the chromatin fraction of control and WRNp-deficient cells. C_V and WRNp-deficient cells (clone 61) were treated with 10 µM RA for 3 days. Chromatin fraction was then separated and both chromatin-lacking lysates (L) and the chromatin fraction (Ch) were analyzed by western blotting. H3 – histone H3, loading control for the chromatin fraction, Trim5α – cytoplasmic fraction loading control. (B) Dnmt3b is present in WRNp immunoprecipitates. Top: WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin-lacking lysates and from the chromatin fraction of undifferentiated and RA treated cells. WRNp immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), D3 – chromatin input lysate immunoprecipitated with the Dntm3b antibody, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. Bottom: Control cells and WRNp-deficient cells (clone 61) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin fraction of undifferentiated and RA treated cells. (C) WRNp is present in Dnmt3b immunoprecipitates. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp and Dnmt3b then were immunoprecipitated from the chromatin fraction. Immunoprecipitates were resolved on SDS-PAGE and WRNp was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), W – lysates immunoprecipitated with a WRNp antibody, D3 - lysates immunoprecipitated with a Dnmt3b antibody, I – lysates immunoprecipitated with normal rabbit IgG. (D) Effect of EtBr and DNAse on Dnmt3b and WRNp co-immunoprecipitation. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp was immunoprecipitated from the chromatin fraction as in Fig. 6B and C, except cell lysates were treated with EtBr (100 µg/ml) or DNAse prior to immunoprecipitation (see Methods). Immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. Et – EtBr-treated lysates, Ds – DNAse-treated lysates, M – mock-treated lysates, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. (E) Q-ChIP analysis of Dnmt3b levels at the Oct4 promoter regions of control and WRNp-deficient cells. Cells were treated with RA for three days as described above and then subjected to Q-ChIP analysis using a Dnmt3b antibody.