Identification of Replisome-associated Proteins by iPOND

iPOND was performed as described previously^{20 (link),38 (link),63} . Briefly, six 15 cm dishes of HCT116 cells were pulse-labeled for 10 min with 10 μM EdU and then either collected directly or washed and treated with 10 μM thymidine for 1 h or 2 mM HU for 3 h. Cells were then cross-linked in 1% formaldehyde in PBS for 20 min at RT, and cross-linking reactions were quenched using 1 mL of 1.25 M glycine. Following permeabilization in 0.25% Triton X-100 for 30 min at RT, cells were washed and resuspended in click reaction buffer (2 mM CuSO₄, 10 mM sodium ascorbate, and 10 μM biotin azide (Invitrogen) in PBS) for 2 h. Cell pellets were washed, lysed in SDS lysis buffer (50 mM Tris (pH 8.0), 1% SDS with protease inhibitor tablet (Roche)), and sonicated for 20 s at 40% amplitude for a total of 5 pulses. Insoluble material was removed by centrifugation, and lysates were transferred to a new tube and diluted 1:1 with PBS. Lysates were incubated for 3 h at 4 °C with 25 μl of streptavidin agarose beads (EMD Millipore). Beads were washed twice with cold lysis buffer, once with 1 M NaCl, and twice more with cold lysis buffer, prior to elution in 2X SDS loading buffer (5% SDS, 25% glycerol, 150 mM Tris (pH 6.8), 200 mM DTT) for 25 min at 95 °C. Subsequent SDS-PAGE and immunoblotting were performed as previously described.