performed using the same microscopy as used in laser micro-irradiation. Immunoblotting Cells were sonicated in lysis buﬀer (50 mM Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% NP-40, 1 mM EDTA) supple- mented with protease inhibitor (complete, EDTA-free, Roche) and cleared by centrifugation. Proteins were separated by SDS/PAGE, electroblotted and detected with the following antibodies: mouse anti-PHF1 (1:1000; M01, Abnova), mouse anti-Ku70 (1:3000; N3H10, Sigma), mouse anti-FLAG (1:3000; F-3165, Sigma), goat anti-actin (1:2000; I-19, Santa Cruz Biotechnology), mouse anti-P53 (1:1000; DO-1, Santa Cruz Biotechnology), rabbit anti-RbAP46 (1:1000; PA1-868, ABR) and rabbit anti-SMC1 (1:1000; BL308, Bethyl). The secondary antibodies used were from Santa Cruz Biotechnology: anti-rabbit IgG-HRP (1:3000, sc-2004), anti-mouse IgG-HRP (1:3000, sc-2005) and anti-goat IgG- HRP (1:3000, sc-2056). Stable cell lines Stable isogenic cell lines expressing either PHF1 or Ku80 tagged with a FLAG-HA were established using Flp-In system (Invitrogen), according to the manufacturer’s protocol. Brieﬂy, we modiﬁed pcDNA5/FRT vector (Invitrogen) to introduce various cDNAs attached with an in-frame XhoI site at the start and NotI site at the stop codons and put two diﬀerent epitope tags in tandem (FLAG-HA) on the 50-terminus of cloning site. Flp-In-293 cells were cotransfected with a 1:9 ratio of pcDNA/ FRT:pOG44 and selected with hygromycin. Hygromycin- resistant cell clones were picked up and expanded. Target protein expression was veriﬁed by immunoblotting. Control cell line was generated by transfecting Flp-In- 293 cells with pcDNA5/FRT blank vector containing Flag-HA tags. To generate V15B cells stably expressing Ku80, V15B cells were transfected with plasmid of pcDNA3.1-His-Ku80 using fugene6. The transfectants were selected by zeocin (500 mg/ml) and conﬁrmed by immunoblotting using anti-Ku80 antibody. Immunoprecipitation and nanoLC/MS/MS Cells were sonicated in lysis buﬀer (50 mM Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% NP-40, 1 mM EDTA) supple- mented with protease inhibitor (complete, EDTA-free, Roche). Lysates were incubated on ice in the presence of 20 mg/ml ethidium bromide (EtBr) for 1 h and cleared by centrifugation, and then the supernatants were collected. Immunoprecipitation was carried out with anti-FLAG M2 aﬃnity gel (A2220, Sigma), according to the manufacturer’s protocol. Proteins were eluted by lysis buﬀer containing 100 mg/ml FLAG peptide (F3290, Sigma). Eluted proteins were detected by immunoblotting. For mass spectrometry, a large-scale suspension culture was carried out. Cells were grown in Joklik medium (Sigma) to 1 l kept between 2 and 6 105/ml. cells were collected and lysated for immunoprecipitation. Eluted proteins were separated on a 12.5% polyacrylamide gel and stained using a Wako Mass silver stain kit. Nucleic Acids Research, 2008, Vol. 36, No. 9 2941 Gel slippage was reduced by 100 mM DTT and alkylated by 100 mM idoacetamide. After washing, the gels were incubated with trypsin overnight at 308C. Recovered peptides were desalted by Ziptip c18 (Millipore). Samples were analyzed by nanoLC/MS/MS systems (DiNa HPLC system KYA TECH Corporation/QSTAR XL Applied Biosystems). Mass data acquisitions were piloted by Mascot software (30). Generation of a stable PHF1 knockdown cell line and colony formation assay Short hairpin siRNA constructs with 21 ribo-nucleotide sequences from PHF1. Oligo-ribo-nucleotides were synthesized and cloned into the psiRNA-h7SKzeo G1 vector (Invivogen). The PHF1 siRNA vector and control vector (psiRNA-h7SKz-Luc, Invivogen) were transfected into HeLa cells by fuGene6 (Roche). Stable transfectants were selected in the presence of 500 mg/ml zeocin. Knockdown of PHF1 was detected by immunoblotting and real-time PCR. Among the siRNA constructs only one sequence (122-GGACTGATGGGCTGCTATACT) had an eﬀect on the expression of PHF1 in HeLa cell. For the colony formation assay, cells were plated in duplicate at 400 cells/6 cm dishes. Eight hours after plating, cells were irradiated with X-ray. Eight days later, colonies were ﬁxed and stained with 0.3% crystal violet in methanol for counting. Three independent experiments were carried out and the standard errors were indicated with an error bar. Recombination assay Recombination assay was carried out as described previously (29). The pCMV3nls-I-sceI expression vector and the HeLa cell line, which contain a stably integrated copy of recombination reporter vector SCneo, were kind gifts from Dr Maria Jasin. Double strand siRNA for PHF1 and scramble were synthesized and puriﬁed by a Silencer siRNA construction kit (Ambion) (PHF1: 50-AA GCTTTCTCTGCCATATGGA. Scramble: 50-AAGCT TACCGTCTCTTAACGA). The HeLa cells in 3.5 cm dishes were transfected with 2 nM siRNA for PHF1 and control by using OligofectAMINE (Invitrogen). The cells were trypsinized and counted 48 h after transfection. With 2 mg pCMV3nls-I-SceI 1 106 cells were electroporated according to manufacturer’s protocol (Digital Bio. 950 V, 35 ms and 2 pulse), and cells were plated in 10 cm dishes. Cells were selected in 1 mg/ml of G418 beginning 24 h after electroporation. The cloning eﬃciency was determined by plating 1000 cells in 10 cm dishes. Colonies were ﬁxed and stained with 0.3% crystal violet in methanol for counting. Three independent experiments were carried out and the standard errors were indicated by an error bar. Real-time PCR Total RNA was extracted from cells by using a High Pure RNA Isolation Kit (Roche). cDNA were synthesized by using a First Strand cDNA synthesis Kit (Roche). Real- time PCR was performed in triplicate using Thermal Cycler Dice Real Time System (TaKaRa). The primer of PHF1 used in quantitative PCR were: 50-TTACTGTTACT GTGGTGGCC, 50-GGTGATACAGGACAAGATGG.