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. 2013 Oct 15;27(20):2259–2273. doi: 10.1101/gad.223180.113

Figure 2.

Figure 2.

Loss of replisome proteins from chromatin in ATR-deficient cells. (A) Western blot detection of total cellular and chromatin-bound proteins as indicated. (B) Western blot detection of SUMO2/3 in chromatin lysates and quantification by optical density. APH was added to the cells as indicated, and chromatin-containing fractions were Western-blotted and detected for SUMO2/3. Note that AtrΔ/− cells consistently exhibited reduced SUMO2/3-conjugated proteins in chromatin lysates after 6 h of APH treatment. (C) Western blot detection of SUMO2/3 in chromatin lysates from RNF4-suppressed cells. This experiment was performed similarly to that described in B, with the exception that RNF4 levels were suppressed by shRNA targeting. Note that while the amplitude of SUMO2/3 presence was variable using crude nuclear-insoluble preparations, the changes shown represent the average effects of the experiments (n = 14). RNF4 suppression prevents the loss of SUMO2/3 conjugates on chromatin in AtrΔ/− cells. (D) Western blot detection of PCNA in chromatin lysates from the indicated cells treated with MG132 or left untreated. Loss of PCNA in AtrΔ/− cells was consistently mitigated by the addition of MG132.