Figure 5

PHRF1 interacts with NBS1 via SDTE motif. (a) HeLa cells were transfected with empty FLAG vector and FLAG-NBS1 construct. Cell extracts were immunoprecipitated (IP) with anti-FLAG agarose and immunoblotted (IB) with anti-PHRF1 antibody. Reciprocal transfection with empty hemagglutinin (HA) vector and HA-PHRF1 was conducted. Input, the same amount of cell extracts for immunoprecipitation. (b) HeLa cells were exposed to CPT (10 μM) for 2 h and then simultaneously labeled with a mixture of anti-PHRF1 mAb (red) and anti-NBS1 polyclonal antibody (green). (c) Cell extracts harvested from empty HA vector-, HA-PHRF1-, HA-PHRF1^S925A-, HA-PHRF1^S925A/S1389A- and HA-PHRF1^S915A/T917A-transfected HEK293T cells were IP with anti-HA agarose and IB with indicated antibodies. (d) Synthetic biotin-labeled S⁹¹⁵DT⁹¹⁷E, A⁹¹⁵DA⁹¹⁷E, pS⁹¹⁵DTE, SDpT⁹¹⁷E, and pS⁹¹⁵DpT⁹¹⁷E peptides (a.a. residues 908–924 of PHRF1) were incubated with HeLa cell extracts. IB analysis was carried out using anti-PHRF1 antibody. (e) ChIP was performed by anti-NBS1 antibody to detect the region of 0.3 kb downstream of I-SceI site. Control IgG for ChIP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for PCR were as controls. (f) Empty HA vector, HA-PHRF1, HA-PHRF1^S925A, HA-PHRF1^S925A/S1389A, PHRF1^S915A/T917A, and HA-PHRF1^C186A/C189A constructs were transfected into NHEJ reporter H1299 cells. The proportion of enhanced green fluorescent protein (EGFP)-positive cells was determined by flow cytometry at 48 h after I-SceI transfection. Each experiment represented the mean±S.D. of three independent experiments. *P<0.05 and **P<0.01 compared with the control