Figure 2.

BRCA2 T77 Is Phosphorylated In Vivo in a CDK-Dependent Manner

(A) Recombinant WT and T77A His-BRCA2 NTD were phosphorylated in vitro with recombinant CDK2/cyclin A, and phosphorylation at T77 was assessed using pT77 phospho-specific antibody.

(B) Western blot analysis of partially purified FE-BRCA2 WT, T77A, and A75P variants using pT77 antibody.

(C) Schematic diagram of full-length BRCA2 showing mutations in Capan-1 and EUFA423 cells indicated by dotted and solid arrows, respectively.

(D) Detection of pT77-BRCA2 in WCEs of HeLa, EUFA423, and Capan-1.

(E) Detection of pT77-BRCA2 after treatment of HeLa cells with CDK inhibitors, 10 μM RO-3306, 60 μM NU6102, or DMSO for 4 hr. Relative signal intensities of pT77 in each sample were quantified against the BRCA2 blot and are indicated under each lane.

(F) Detection of pT77-BRCA2 in synchronized HeLa cells using double thymidine block and release. Rad51 complexes were immunoprecipitated from the soluble fraction and analyzed using the indicated antibodies. To monitor progression through the cell cycle, chromatin-bound PCNA (S phase) (Moldovan et al., 2007) and Histone H3 phosphorylated at Serine 10 (pS10-H3; mitosis) (Hans and Dimitrov, 2001) were detected. The asterisk indicates a nonspecific band.

(G) Cell-cycle distribution analysis of propidium-iodide-stained cells. As, asynchronous cells; Noc, nocodazole-treated cells.

See also Figure S2.