Figure 3.

BRCA2 Facilitates Plk1-Dependent Rad51 Phosphorylation at S14

(A) HEK293T cells were treated with IR (4 Gy, 20 min recovery), and pS14-Rad51 from the indicated cellular fractions was immunoprecipitated with pS14 antibody and detected with Rad51 antibody. Cyt, cytoplasmic soluble fraction; Nuc, nuclear soluble fraction; Chr, chromatin-bound fraction.

(B) Schematic diagram of the BRCA2 alleles in DLD1 BRCA2^−/− and BRCA2^+/− cells.

(C) Endogenous Rad51 was immunoprecipitated from DLD1 BRCA2^−/− or BRCA2^+/− cells before and after irradiation (4 Gy). Thirty minutes after IR treatment, nuclear soluble fraction was prepared and pS14 was detected as in Figure 3A.

(D) Schematic diagram of FE-BRCA2 variants as shown in Figure 1E. Amino acid substitutions within the BRC (red asterisks and bars) and TR2 motifs (blue asterisk and bar) that impair Rad51 interaction are also indicated.

(E) FE-tag, FE-BRCA2 WT, BRC, TR2, or BRC/TR2 variants were conditionally expressed in HEK293 Flp-In T-REx cells and the level of S14 phosphorylation was assessed by IP with pS14-Rad51 antibody.

(F) As in Figure 3E, BRCA2 variants were conditionally expressed in HEK293 Flp-In T-REx cells, and the level of S14 phosphorylation and association of BRCA2 and Plk1 were assessed following Rad51 IP.

See also Figure S3.