Enhanced enrichment of RECQ1 at lamin B2 origin after replication stress. A. Genomic regions containing the human lamin B2 origin of replication. Primer sets used for qPCR analyses of origin (B48) and non-origin (B13) containing DNA are indicated. B. Quantification of cross-linked lamin B2 origin immunoprecipitated using the indicated antibodies. HeLa cells were either untreated or incubated with APH (0.5 μM) or HU (2 mM) for 24 h and processed for ChIP using a RECQ1-specific antibody. ORC2 antibody was used as positive control for origin-enrichment, and rabbit IgG served as negative control. qPCR was performed with origin-specific and non-origin primers for the lamin B2 locus. Fold enrichment was determined over IgG and is shown for each primer pair. Results are expressed as means ± SEM for at least three independent experiments. Specificity of RECQ1 antibody in immunoprecipitation (IP) of endogenous RECQ1 from HeLa extract is shown; input represents 10% of the extract used for IP. C . A representative gel of the amplified DNA immunoprecipitated with indicated antibodies. Input represents 1% of the cross-linked chromatin used for ChIP. D . Cell cycle profiles of untreated and HU or APH treated (24 h) HeLa cells. Propidium iodide stained cells were analyzed by FACS for DNA content; distribution in the G1, S, and G2/M phases of the cell cycle is indicated. E . Total RECQ1 protein level in HeLa cells following 24 h treatment with HU (2 mM) or APH (0.5 μM). GAPDH is loading control. F . Replication stress-dependent chromatin association of RECQ1. Western blot detection of RECQ1 in chromatin-enriched fractions of untreated and APH (0.5 μM), HU (2 mM) or MMC (0.5 μg/ml) treated cells, or following pretreatment with APH for 4 h before incubation with MMC (16 h). Histone H3 is loading control and chromatin marker. APH, aphidicolin; HU, hydroxyurea; MMC, mitomycin C.