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. 2012 Aug 10;287(40):33327–33338. doi: 10.1074/jbc.M112.357996

FIGURE 6.

FIGURE 6.

Association of DNA polymerases with lamins A/C in S phase. HeLa cells were synchronized by double thymidine block to early S phase (0 h) and released to proceed in S phase (6 h). A, co-precipitation of lamin A/C in chromatin immunoprecipitates of Pol α, δ, and ϵ. B, co-precipitation of replicative DNA polymerases in lamin A/C chromatin immunoprecipitates. The antibody used for immunoprecipitation is indicated on top and the antibody used for Western blotting on left side of the panels. Nucleoproteins for chromatin immunoprecipitation were isolated and treated as described under “Experimental Procedures.” Nucleoproteins derived from CsCl centrifugation were sonicated and digested with micrococcal nuclease as described under “Experimental Procedures.” Soluble nucleoproproteins, 1 mg/500 μl, were taken for immunoprecipitation with the cognate antibodies. 10 μl of soluble nucleoprotein complex (In), 10 μl of supernatant after precipitation (S), and the entire precipitate (P) were loaded onto the gel. C, quantification of the lamin A/C in chromatin immunoprecipitates with antibodies against Pol α, δ, and ϵ, respectively. The lamin A/C signal in the precipitate of the cognate DNA polymerase was quantified relative to the signal in the input on the same blot. The values represent the mean ± S.D. of two to four independent experiments. D, association of LB2 origin DNA with lamins A/C at 0 and 6 h after release from double thymidine arrest in nucleoprotein complexes. Nucleoproteins derived from CsCl centrifugation were sonicated and digested with micrococcal nuclease as described under “Experimental Procedures.” Soluble nucleoproproteins, 1 mg/500 μl, were taken for immunoprecipitation with the cognate antibodies, the protein was digested with proteinase K, and DNA isolated. 5 to 10 ng of DNA were obtained from the immunoprecipitate, 1/20 was used for quantitative PCR (precipitate). DNA from soluble nucleoproteins representing genomic DNA was isolated and treated in the similar manner, except that the immunoprecipitation step was omitted, and analyzed by quantitative PCR (input). The values are mean values from two distinct experiments.