Anti tlr9

To investigate the role of TLR9 in MWCNT-induced DNA damage, A549 cells were transfected with 10 nM TLR9 siRNA (s28872 or s28873, Silencer® Select, Ambion) by using Lipofectamine® RNAiMAX reagent (Invitrogen) in Opti-MEM® I medium (Gibco), and incubated for 2 days at 37 °C. To exclude the possibility of off-target effects, cells were transfected with 10 nM Negative Control #2 siRNA (Ambion) instead. Depletion of TLR9 expression by siRNA was confirmed by fluorescent immunocytochemistry as described above and Western blotting as described below. In Western blotting, we used anti-TLR9 (1 μg/ml, Santa Cruz Biotechnology, sc-25468) and anti-GAPDH (0.2 μg/ml, Santa Cruz Biotechnology, sc-25778) rabbit polyclonal antibodies as primary antibodies and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG antibody (0.2 μg/ml, Santa Cruz Biotechnology) as the secondary antibody. Quantitative image analysis was performed with an ImageJ software.

LPS and CpG-ODN were purchased from Invivogen. DNase I and RNase were purchased from Ambion and Invitrogen, respectively. α-GalCer was purchased from Funakoshi. Mouse Flt3L were purchased from Peprotech. Anti-Flag-tag (SIGMA), anti-cMyc-tag (Clontech), anti-TLR9 (Santa Cruz) and anti-GzmA (rabbit polyclonal) (LSbio) antibodies were from the indicated suppliers. Anti-CD11c (HL3), -CD40 (3/23), -CD80 (6-10A1) and -IFN-γ were purchased from BD. Anti-CD8 (53.67), -CD45.1 (A20), -CD86 (GL-1), -Siglec H(551) and Annexin-V-APC were purchased from Biolegend. Anti-B220 (RA3-6B2) was purchased from e-Bioscience. OVA-tetramers were purchased from MBL. CFSE and Mito-tracker were purchased from Molecular Probes. OVA_257−264 peptide (SIINFEKL) was obtained from Toray Research Center, Inc. Chloropromazine and Latrunculin A were purchased from Wako Co. Diphtheria toxin were purchased from Sigma.