Fluorescein fitc conjugated goat anti mouse igg

LPS was purchased from Sigma (St. Louis, MO, USA). Primary antibodies against CXCR4 (40kDa, ab80791), CXCR7 (42kDa, ab72100) and TLR4 (92kDa, ab30667) were purchased from Abcam Ltd. (Cambridge, UK). Rabbit anti-human antibodies against p38 MAPK (43kDa), phospho-p38 MAPK (43kDa), ERK1/2 (42/44kDa), phospho-ERK1/2 (42/44kDa), JNK (54kDa), phosphor-JNK (54kDa), NF-κB p65 (65kDa), phosphor-IKKɑ/β (85/87kDa), MEK1/2 inhibitor U0126 and JNK inhibitor SP600125 were purchased from Cell Signal Technology (Beverly, MA, USA). Rabbit polyclonal IκBɑ (41kDa), IKKɑ/β (85/87kDa) and phosphor-IκBɑ (41kDa) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated monoclonal mouse anti-GAPDH antibody was purchased from Zen BioScience (Chengdu, China). Fluorescein (FITC)-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The NF-κB inhibitor BAY 11–7082 was obtained from Beyotime Institute of Biotechnology (Shanghai, China). The BCA protein assay kit and enhanced chemiluminescence (ECL) detection system were purchased from KeyGEN (Nanjing, China).

Immunfluorescence detection of phospho- H2AX and RAD51 foci was performed to monitor DNA double-strand breaks (DSBs) formation and homologous recombination repair (HRR). Cells cultured on coverslips were treated with 10 nM YM155 and irradiated with a dose of 4 Gy to assure a discrimination of individual nuclear foci in immunofluorescence staining. At indicated time points, the cells were fixed by 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. After blocking with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 1 h at room temperature, cells were incubated with antibody for phospho-H2AX (Ser139) (Millipore/Upstate, Temecula, CA) and RAD51 (EMD Millipore, Billerica, MA) at 4°C overnight, followed by staining with Fluorescein (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch, PA) and Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, PA) for 1.5 h at room temperature. Finally, the samples were counterstained with 2 μg/ml DAPI and mounted in 3 μl of mounting medium (Beyotime). Three random fields each containing 50 cells were examined at a magnification of ×100 under a Zeiss LSM5 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). Nuclei containing ≥10 immunoreactive foci were scored as positive for γ-H2AX, and ≥5 foci as positive for RAD51.