Figure 5.

S14 Is Important for Rad51 Accumulation at Replication Forks

(A) Depiction of the iPOND experimental procedure.

(B) Proteins associated with replication fork in HEK293T cells with or without 4 mM of HU treatment were isolated by iPOND procedure, and detected with indicated antibodies. In order to discriminate proteins associated with DNA behind replication forks, EdU-labeled cells were subsequently incubated with media containing a low concentration of thymidine (10 μM), and associated proteins were similarly detected (thymidine chase).

(C) HEK293T cells were pretreated with 100 nM of Plk1 inhibitor BI 2536 for 30 min, followed by HU treatment and iPOND analysis as in (B). Right panel shows the quantitation of one representative iPOND experiment, expressed as the relative ratio of the total level of Rad51 against H2A.X.

(D) HEK293 Flp-In T-REx cells conditionally expressing FLAG-tagged Rad51 WT or S14A, along with shRNA targeting the 3′ UTR of endogenous RAD51, were treated with HU, and proteins associated at nascent DNA were analyzed as in (B).

(E) As in (D), except that cells were pretreated with BI 2536 for 30 min as in (C).

(F) As in (D), except that endogenous BRCA2 rather than Rad51 was downregulated by shRNA.

See also Figure S5.