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. 2023 Feb 6;6(4):e202101186. doi: 10.26508/lsa.202101186

Figure 4. iPOND protein recovery is biased by replication organization.

Figure 4.

(A) Experimental setup. HeLa S3 cells were submitted to the thymidine block for 18 h and released into the S phase. Cells were collected at T0 (G1), T2 (Early-S), T4 (Mid-S), and T8 (Late-S) after a 15-min EdU pulse for iPOND and flow cytometry. Replication patterns showing the different phases are represented. (B) The percentage of cells in each phase was analyzed using flow cytometry. The error bars represent the variations within three independent experiments. (C) iPOND experiment performed on unsynchronized and synchronized cells and analyzed by Western blot using antibodies directed against the indicated proteins. In no-click samples, biotin–TEG azide was replaced by DMSO. (D) Quantification of the indicated proteins in iPOND based on at least three independent experiments; T0 was used for normalization.