Phospho kap1

Fibroblasts were transfected with 200 nM of ASOs as described above. Forty-eight hours after transfection, cells were irradiated with 1.5 Gy using a caesium-137 source, and then incubated for 60 min at 37 °C. Cells were washed in PBS, fixed in 4% (w/v) paraformaldehyde and permeabilized with 0.1% (w/v) Triton X-100 in PBS at room temperature. Cells were then incubated overnight in PBS with 3% BSA and antibodies to phospho-P53 (Cell Signaling Tech) and phospho-KAP1 (Bethyl Lab) and were visualized with immunoglobulin G Alexa Fluor conjugates (Life Technologies). DNA was counterstained with Hoechst 33342. Images were collected with the ImageXpress Micro microscope (Molecular Devices) and processed with MetaXpress (Molecular Devices). The abundance of targets expressed in nuclei was quantified.

Immunostaining was performed essentially as in ref. [14 (link)] and ref. [30 (link)]. Cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized with 0.5% NP-40 and blocked in 3% BSA.The following primary antibodies were used: UBF (H-300, Santa Cruz Biotechnology), NCL (4E2, Abcam), RPA194 (C-1, Santa Cruz Biotechnology), phospho-ATM (Cell Signaling Technology), γH2AX (Millipore), phospho-KAP1 (Bethyl Laboratories), phospho-DNA-PKcs (Abcam). Secondary Alexa488 and Alexa594-cojugated anti-mouse and anti-rabbit antibodies were from Invitrogen. DNA was stained with DAPI. Images were captured using Axioplan2 fluorescence microscope (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar 20x/0.5 and 40x/0.75 objectives (Zeiss). Image analysis was conducted using FrIDA designed for the analysis of RGB color image datasets as in ref. [14 (link)] and ref. [25 (link)]. Hue saturation and brightness ranges for green and red fluorescence channel and DNA (blue) were defined for each image set. Image intensities were determined as the fraction of positive cells divided total nuclear area as defined by DNA staining. An average of 100 cells was quantified from two fields for each sample.

Protein extracts were separated on SDS–PAGE and were transferred to a PVDF membrane (IPVH00010; Millipore). SuperSignal® West Pico Chemiluminescent substrate (34080; Thermo) was used to visualize the signal. The membranes were reprobed after incubation in stripping buffer (21059; Thermo). Antibodies against the following proteins were used: p65 (sc-372; Santacruz), RelB (4922; Cell signaling), c-Rel (sc-71; Santacruz), Tubulin (T6074; Sigma), Phospho-KAP-1 (A300-767A; Bethyl) and p53 (2524; Cell signalling).