5hh2 2a8

J774A.1 cells, cultured on glass-based dishes, were preincubated with anti CD16/CD32 mAb (93, Biolegend) to block the FcγRII/III receptors and incubated with anti-histone H2B mAb (5HH2-2A8, Millipore, 1:200 dilution) and either 100 µg/ml Bt-BSA or Bt-AGEs at 4 °C for 15 min in HBSS-BSA. After washing three times with PBS (−), the cells were stained with streptavidin-Alexa Fluor 568 conjugate (1:1,000 dilution, Invitrogen) and anti-mouse IgG-Alexa Fluor 488 conjugate (1:1,000 dilution, Invitrogen) at 4 °C for 15 min in HBSS-BSA. After washing, the stained cells were fixed with 4% paraformaldehyde in PBS (−) for 10 min at room temperature and further stained with 1 µg/ml 4’,6-Diamidino-2-phenylindole (DAPI, Dojindo). Fluorescent images were obtained using a confocal microscope (FV1200, Olympus) and analyzed by Olympus software (FV10-ASW, Olympus).

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.