Rabbit pab anti hmgb1

Western blot analysis was performed as previously reported (40 (link)). Expression levels of HMGB1 were evaluated by using the Rabbit pAb anti-HMGB1 (Abcam Ltd). Actin signal was detected by Mouse mAb anti-β-actin (Sigma Aldrich) and used as loading control.

Formaldehyde-cross-linked chromatin fragments obtained from HeLa cervical cancer cells were immunoprecipitated with Rabbit pAb anti-HMGB1 (Abcam Ltd.) and with Rabbit pAb anti-TRF1 N19 (Santa Cruz Biotechnology) as positive control for telomeric sequences. Chromatin fragments immunoprecipitated without the antibody (No Ab) and with Normal Rabbit immunoglobulins (IgG) (Santa Cruz Biotechnology) were used as negative controls of the ChIP assay. After precipitation, the assay was performed as previously described (41 (link)).

Cells were fixed in 2% formaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature (RT) and permeabilized in 0.25% Triton X-100 in PBS for 5 min at RT. For immune-labeling, cells were incubated with primary antibody for 2 h at RT, washed twice in PBS and finally incubated with the secondary antibodies for 1 h. The following primary antibodies were used: Rabbit pAb anti-HMGB1 (Abcam Ltd, Cambridge, UK), Mouse mAb anti-γH2AX (Millipore, Billerica, MA, USA) and Rabbit pAb anti-TRF1 N19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following secondary antibodies were used: Anti-Mouse IgG (H+L), F(ab’)₂ Fragment (Alexa Fluor 488 Conjugate) (Cell Signaling) and Anti-rabbit IgG (H+L), F(ab’)₂ Fragment (Alexa Fluor 555 Conjugate) (Cell Signaling). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Solms, Germany). For quantitative analysis of γH2AX positivity, 300 cells on triplicate slices were scored and for TIF analysis, 30 γH2AX-positive cells were scored. Cells with at least four co-localizations (γH2AX/TRF1) were considered as TIF-positive. Where reported, cells were incubated with the indicated doses of Braco-19 for 24 h.