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. 2015 Sep 9;43(20):9835–9855. doi: 10.1093/nar/gkv880

Figure 1.

Figure 1.

RAD51 paralogs associate with S-phase chromatin and bind to nascent DNA at stalled/collapsed replication forks. (A) U2OS cells were synchronized by serum starvation (G0/G1), thymidine-aphidicolin block (S) and nocodazole (G2/M). Chromatin extracts were prepared at indicated cell cycle stages and analyzed for RAD51 and RAD51 paralogs. (B) Quantification of western blots from two independent experiments showing fold enrichment of indicated proteins at the chromatin at indicated cell cycle stages. (C) Experimental design for nascent strand pull-down of active replication forks. HeLa cells were labeled with IdU for 30 min, followed by a chase into thymidine-containing medium for the indicated times. Cells were cross-linked, and the chromatin fraction was isolated (input) and subjected to IP using anti-IdU antibody (Idu-IP) or only beads (Beads control). Fractions were probed for indicated proteins with ORC2 as control (input and negative control). (D) Experimental design for nascent strand pull-down of HU-mediated stalled/collapsed replication forks. HeLa cells were labeled with IdU for 30 min, followed by a chase into 1 mM HU-containing medium for the indicated times. Representative neutral comet images of HeLa cells treated with 1 mM HU for the indicated times are shown. Cells were cross-linked, and the chromatin fraction was isolated and subjected to IP using anti-IdU antibody (Idu-IP). Fractions were probed for indicated proteins with Histone H3 as loading control.