Rabbit anti cenpf

Immunofluorescence experiments were carried out as described previously.^{14 (link)} Briefly, A549 cells were γ irradiated (2 Gy) and cultured at 37 °C for the indicated periods of time. The cells were then rinsed in PBS and fixed in 4% paraformaldehyde for 5 min. Cells were subsequently permeabilized in 0.2% Triton X-100 for 2 min, blocked with 5% bovine serum albumin for 30 min, and incubated with mouse anti-phospho-histone γH2AX (Ser139) antibody (Millipore, 1:1000 dilution) and rabbit anti-CENPF (Abcam, 1:500) at 4 °C overnight. Cells were washed three times with PBS containing 0.1% Tween 20 and 0.02% SDS and incubated with Alexa Fluor 488 goat antimouse and Alexa Fluor 555 goat antirabbit secondary antibodies (Invitrogen, 1:500 dilution) at room temperature for 1 h. After washing the cells five times as described above, they were stained with DAPI (Sigma), mounted in Vectashield Mounting Medium (Vector Laboratories), and scored for γH2AX foci in G1 (CENPF-negative) cells using a Nikon Eclipse 50i microscope as described previously.^{14 (link)}