Rabbit anti phospho histone h3 ser10 antibody

The slides were submerged in 10 mM citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 30 min at 95°C, incubated for 30 min with a blocking solution (PBS pH 7.2, 0.05% Tween 20, 0.1% Triton X-100, 2% goat serum, 2% BSA) in a humid chamber at RT, then incubated overnight at 4°C with rabbit anti-phospho-histone H3 (Ser10) antibody (Cell Signaling Technology, Danvers, MA) diluted 1/800 in PBS, washed three times for 2 min with washing solution (PBS pH 7.2, 0.05% Tween 20) and then incubated for 30 min at RT with the fluorescein-labeling goat anti-rabbit antibody (Jackson ImmunoResearch Lab, West Grove, PA) diluted 1/500 in PBS. The sections were then washed with PBS and mounted with VectaShield mounting medium with DAPI (Vector, Laboratories, Burlingame, Ca). The human cell line U251 was included as a proliferation control. Images were captured by confocal microscopy (Zeiss, LSM880, Oberkochen, Germany).

The phosphorylation of histone H3 by PirB was determined using Western blot analysis by treating primary shrimp hemocytes with rPirB. Briefly, hemocytes were collected and cultured, followed by treatment with different concentrations of His-rPirB or His-rPirA (0.2 µM or 0.8 µM) at 28°C for 2 h. Next, cells were lysed for 10 min at room temperature with lysis buffer (containing 25 mM HEPES, 150 mM NaCl, 1 mM EDTA-Na₂ · 2H₂O, 1% Triton X-100, 0.5% SDS and 1× Protease Inhibitor Cocktail Tablets, Roche, Switzerland) before being centrifuged at 20,000 g for 30 min at 15°C to collect the supernatant. The collected supernatant was analyzed using SDS-PAGE and Western blot, as described in subsection 2.2, with mouse anti-histone H3 antibody (1:1000, Transgen BioTech, Beijing, China), and rabbit anti-phospho histone H3 (Ser10) antibody (1:1000, Cell Signaling Technology, MA, USA).