Anti fanci

For immunofluorescence (IF) analyses, cells were seeded in four-well tissue culture slides (BD Biosciences) or on cover slips (Corning) and treated with MMC for 18 h. Cells were fixed in 4% w/v paraformaldehyde in PBS for 15 min on ice, followed by permeabilization for 5 min in 0.3% v/v Triton X-100 in PBS. Fixed cells were incubated with primary antibodies in 5% v/v goat serum, 0.1% v/v NP40, in PBS for 1 h, washed three times with PBS and then incubated with Alexafluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen) for 45 min. Cells were then counterstained and mounted in vectashield plus 406-diamidine-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories) and visualized using a Zeiss AxioImager.A1 upright epifluorescence microscope with AxioVision LE 4.6 image acquisition software. Primary antibodies used for IF were anti-53BP1 (H300; Santa Cruz Biotechnology), anti-DNA-PKcs pS2056 (ab18192; Abcam), anti-FANCA (ABP6201; Cascade), anti-FANCD2 (NB100–182; Novus Biologicals and sc-20022; Santa Cruz Biotechnology), anti-FANCI (A300-212A; Bethyl Laboratories), anti-FANCM (Meetei and Deans CE56.1 antibodies), anti-FK2 (sc-8017; Santa Cruz Biotechnology), and anti-γH2AX (05–636; Millipore).