Rabbit anti 53bp1 antibody

Cells (1 × 10⁴) were seeded onto four-well chamber slides (Fisher Scientific, Leicestershire, UK) and left overnight to adhere. For PTEN-depleted damage repair studies, cells were irradiated with 1 Gy (to prevent damage saturation) and analysed at indicated time points ranging for 0.5 to 24 h post-radiation to observe repair kinetics. For CXCR1/CXCR2 knockdown experiments, transfections were performed 72 h prior to irradiation. Cells were fixed 4 h post-radiation treatment with 50% methanol:50% acetone, and permeabilized in 0.5% Triton X-100 (Sigma, Dorset, UK). After an overnight incubation in blocking buffer (PBS, 0.1% Triton X-100, 5% FCS, 0.2% milk), cells were incubated with rabbit anti-53BP1 antibody (Abcam, Cambridge, UK) at 1:2000 concentration and incubated with secondary anti-rabbit Alexa Fluor 488 (Life Technologies, Paisley, UK). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (0.1 mg/ml). Foci were counted using a fluorescence microscope (Zeiss Axiovert 200M, UK); typically, 100 cells were counted per treatment condition.

At different times after irradiation, HT22 cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, USA) for 15 min at room temperature (RT). After permeabilization and blocking, the cells were sequentially incubated with primary antibody (rabbit anti-53BP1 antibody (1:200, Abcam, Cambridge, UK) or rabbit anti-LC3 II antibody (1:200, Cell Signaling Technology, Inc., Beverly, MA, USA), and Alexa Flour 488-conjugated goat anti-rabbit secondary antibody (2 μg/ml, Beyotime, China), followed by counter staining with 4′,6′-diamidino-2-phenylindole (DAPI, 5 μg/ml, Beyotime, China). Then the cells were mounted with Antifade Mounting Medium (Beyotime, China) and observed under a fluorescent microscope (Leica DM 2000, Germany). The numbers of 53BP1 foci in at least 500 cells for each sample were scored, the pictures of LC3 II were taken.