Histone h3 1g1

The samples were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was incubated for 1 h with Blocking One (Nacalai Tesque) to block nonspecific binding. The membrane was then incubated overnight at 4 °C with antibodies against histone H2B (5HH2-2A8, Millipore), histone H1.2 (19649-1-AP, Proteintech), histone H2A (938CT5.1.1, Santa Cruz), histone H3 (1G1, Santa Cruz), histone H4 (L64C1, Cell signaling), RAGE (A-9, Santa Cruz), and MMP-9 (AB19016, Millipore) in 10% Blocking One-PBST, followed by the incubation for 1 h at room temperature with HRP-conjugated secondary antibodies (1:5,000 dilution, anti-mouse IgG, Cell signaling, #7076; 1:5,000 dilution, anti-rabbit IgG, Cell signaling, #7074). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and LAS4000 (Cytiva). The data were analyzed by ImageJ Fiji software version 1.52n. For the detection of total proteins, PVDF membrane was stained with 0.1% Ponceau S solution containing 5% acetate for 5 min. The stained membrane was scanned with LAS4000, destained with PBST, and then used for subsequent blocking and immunodetection for histone H2B.

Antibodies were purchased from Cell Signaling Technologies (Danvers, MA) unless otherwise indicated. These include mouse IgG against Caspase 8 (1C12), Histone H3 (1G1) (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc-2 (17) (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology); rabbit IgG against p21, PARP, Cleaved Caspase 3 (Asp175) (5A1E), LC3A/B (D3U4C), α/β tubulin, p-Cdc-2 (T14/Y15) (Santa Cruz Biotechnology), p53 (FL-393) (Santa Cruz Biotechnology), and p-Histone H3 (Ser10) (Upstate Cell Signaling Solutions, Lake Placid, NY). Secondary antibodies used for immunofluorescence include donkey anti-rabbit IgG for Alexa Fluor 594 (Invitrogen, Waltham, MA). Nuclei were visualized by DAPI (Sigma-Aldrich, St. Louis, MO) staining at 1 µg/ml for 5 min.
Western Blot analyses were carried out as previously described [55 (link)]. Western blots were visualized by chemiluminescence using Bio-Rad ChemiDoc™ Imaging System (Hercules, CA) and specific protein signals were quantified using the Fiji-ImageJ analytical software (NIH, Bethesda, MD).