Technical Reference #1942

1942.

APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells Stuart L. Rulten; Felipe Cortes-Ledesma; Liandi Guo; Natasha J. Iles; and Keith W. Caldecott, University of Sussex, Molecular and Cellular Biology, 28(1942), (2008)
Link To Paper

Materials & Methods:
Immunofluorescence. For laser-induced UVA damage wild-type A549 cells or A549-PARP-1KD cells stably depleted of PARP-1 (10) were seeded onto gridded coverslips (MatTek). After preincubation for 10 min with 10 g/ml Hoechst dye 33258 at 37°C selected cells were irradiated by using a 351-nm UVA laser (approximately 0.35 J/m2). Where indicated (see Fig. 1A) cells were preincubated with the PARP inhibitor KU58948 (500 nM). After exposure coverslips were incubated at 37°C for 4 min and washed in ice-cold phosphate-buffered saline (PBS). For H2O2 treatment cells were treated with 10 mM H2O2 in PBS on ice for 20 min rinsed in PBS and incubated for 1 min in drug-free medium at 37°C. Following DNA damage and repair incubation all cells were rinsed in ice-cold PBS and fixed in PBS–4% paraformaldehyde (5 min at room temperature) followed by methanol (20 min at 20°C). Cells were then permeabilized with PBS–1% Triton X-100 (5 min) blocked in PBS–5% nonfat dried milk (NFDM) for 30 min and incubated in 1% NFDM–PBS-TS (0.1% Tween-20 0.02% sodium dodecyl sulfate [SDS] in PBS) containing anti-pADPr monoclonal antibody (MAb) (10H; Alexis) and anti-APLF polyclonal antibody (SK3595 [14]) at 1/200 dilution for 2 h at room temperature. After being rinsed (three times) with PBS-TS cells were incubated in Alexa Fluor 488 goat anti-mouse immunoglobulin G and Alexa Fluor 555 anti-rabbit immunoglobulin G (Invitrogen) at 1/200 dilution in 1% NFDM–PBS-TS for 1 h at room temperature. Cells were then rinsed in PBS-TS (five times) and mounted in Vectashield (Vector Laboratories).

Microscopic Technique
Immunofluorescence Microscopy

Cell Type(s)
wild-type A549 cells or A549-PARP-1KD cells