Gtx115518

A549 cells were treated with 4 Gy IR and whole-cell lysates were isolated at 0 (no IR), 45 min and 3 h after IR and crosslink IP were performed using Pierce Crosslink Immunoprecipitation kit (Pierce) using the manufacture's protocol. Briefly, 10 μg of ATM 2C1 (GTX70103, GeneTex) or CLP1 N3C3 (GTX115518 GeneTex) antibody were incubated with protein AG plus agarose for 60 min at room temperature followed by crosslinking the bound antibody to Protein A/G plus agarose with DSS crosslinker. 1 mg of pre-cleared lysate was added to the antibody-crosslinked resin and incubated overnight at 4 °C. The resin was washed to remove unbound proteins and then immunoprecipitated proteins were eluted using elution buffer. Eluted proteins were analysed by western blotting using anti-ATM Y170 (Genetex GTX61188 at 1:1,000) and anti-CLP1 N3C3 (GeneTex GTX115518 at 1:1,000).

A549 cells in log growth phase were irradiated with 2 Gy. Nuclear and cytoplasmic protein fractions were separated using the NE- PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) at 30 min and 3 h post IR; the 0-h time point (no-IR) was lysed in tandem with the 3 h time point. 50 μg of total protein was boiled in SDS Sample buffer and separated on a 4–20% TGX Criterion Precast Gel (Bio-Rad). Protein was transferred to polyvinylidene difluoride membrane (Millipore) and proteins were probed using the following antibodies: anti-ATM 2C1 (1A1) (ab78, Abcam, 1:1,000), anti-Clp1 N3C3 (GTX115518, GeneTex, 1:1,000), anti-p48 5E10 (ab487, Abcam, 1:1,000), anti-GAPDH 6C5 (ab8245, Abcam, 1:20,000) and sheep anti-mouse IgG, HRP-linked Ab (NA931, GE Healthcare, 1:50,000) and HRP-conjugated anti-rabbit (ab6721, Abcam, 1:25,000). Pierce ECL western blotting substrate (PI-32109) was used for detection.

A549 cells were seeded in a 60-mm plate at 80% confluency. The following day, cells were transfected with 1 μg of plasmid DNA (psi-miR-34 WT or psi-miR-34 MT) and 200 pmol ON-TARGETplus siRNA SmartPool (Dharmacon) using Lipofectamine 2000 as indicated. After transfection, the cells were re-plated in a six-well plate at 50% confluency. After a 12-h incubation, the cells were exposed to 2 Gy of IR. At 4, 12 and 24 h post IR, cells were washed with PBS and lysed in wells with Passive Lysis Buffer (Promega). 25 μl of lysate was withdrawn for luciferase analysis (described above); 75 μl of lysate was boiled in SDS Sample buffer and samples were separated using a 4–20% Criterion TGX Precast Gel (Bio-Rad). Protein was transferred to Whatman BA85 Nitrocellulose and protein was detected using: anti-ATM 2C1 (1A1) (ab78, Abcam, 1:1,000), anti-Clp1 N3C3 (GTX115518, GeneTex, 1:1,000) and sheep anti-mouse IgG, HRP-linked Ab (NA931, GE Healthcare, 1:50,000) and HRP-conjugated anti-rabbit (ab6721, Abcam, 1:25,000). Pierce ECL western blotting substrate (PI-32109) was used for detection.