Rabbit anti 53bp1

DNA DSBs were quantified in spatially (three-dimensionally = 3D) fixed cells by the means of high-resolution ICM detection of co-localized γH2AX and 53BP1 repair foci as described earlier [16 (link),20 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (10 min, at room temperature RT) prior to irradiation (0 min PI, non-irradiated controls) and in several time points PI covering a long (48 h) PI period (5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h PI). Cells were permeabilized in 0.2% Triton X-100/PBS (15 min, RT) and immunoassayed with mouse antiphospho-H2AX (serine 139) (Merck, Darmstadt, Germany, cat. no.: 05-636) and rabbit anti-53BP1 (Cell Signaling Technology, Danvers, MA, USA, cat. no.: 4937) primary antibodies to simultaneously detect the γH2AX and 53BP1. Antiphospho-H2AX antibody was visualized with the secondary FITC-conjugated donkey anti-mouse antibody and anti-53BP1 antibody with Cy3-conjugated donkey anti-rabbit antibody (both Jackson Laboratory, West Grove, PA, USA, cat. no.: 715-095-150 and 711-165-152). Chromatin was counterstained with 1 μM TO-PRO-3 (Molecular Probes, Eugene, OR, USA) prepared in 2× saline sodium citrate (SSC). After brief washing in 2× SSC, Vectashield medium (Vector Laboratories, Burlington, Ontario, Canada) was used for sample mounting.

Cells harvested, pelleted at 1,500 rpm at 4°C for 5 min and lysed with whole cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and cocktail protease inhibitors). Protein concentration was determined using the Bradford assay (Sigma). Protein bands were separated on a NuPAGE 4–12% Bis-Tris Gel (Invitrogen). Proteins were electrotransferred onto a nitrocellulose membrane in 1X NuPAGE transfer buffer. After blocking with 5% skimmed milk solution in 1% Tris-Buffered saline with Tween 20 (TBST) buffer, the membranes were immunoblotted with mouse anti-Flag antibody (Sigma, #F3165, 1:1000), rabbit anti-TDP-43 (Proteintech, #10782-2-AP, 1:1000), mouse anti-phospho-Histone H2.AX (S139) (EMD Millipore, #16-193, 1:1000), rabbit anti-H2.AX (Cell Signaling Tech, #2595, 1:1000), rabbit anti-phospho-ATM (S1981) (Abcam, #ab81292, 1:800), rabbit anti-ATM (Abcam, #ab32420, 1:1000), rabbit anti-phospho-53BP1 (S1778) (Cell Signaling Tech, #2675, 1:1000), rabbit anti-53BP1 (Cell Signaling Tech, #4937, 1:1000), and mouse anti-β-actin (Proteintech, #66009-1-Ig, 1:5000) antibodies. Protein bands were visualized by probing with corresponding HRP-conjugated secondary antibodies and developed with enhanced chemiluminescence reagent in Odyssey (LI-COR).

Naïve CD4 T cells were isolated and cultured as described above, followed by immunofluorescence staining using a method described previously^{6 (link)}. Rabbit anti-53BP1 (Cell Signaling) and mouse TRF1 (Thermo Fisher) were used as primary antibodies and anti-rabbit IgG-Alexa Fluor 488 and anti-mouse IgG- Alexa Fluor 555 (Invitrogen) were used as secondary antibodies. Then, cells were washed and mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). Images were acquired with a confocal laser-scanning inverted microscope (Leica Confocal, Model TCS sp8, Germany).