Cy3 conjugated goat anti mouse igg antibody

A mouse mAb cocktail against CPS1 and P-CK and a rat anti-human CD45 monoclonal antibody (Santa Cruz, CA, USA) were used as primary antibodies. After blocking nonspecific binding sites, slides were incubated for 1 hour with primary antibodies at 37°C. A Cy3-conjugated goat anti-mouse IgG antibody and an Alexa Fluor 488-conjugated rabbit anti-rat IgG antibody (Invitrogen) served as secondary antibodies. After DAPI staining to visualize nuclei, the slides were mounted in antifade solution (Beyotime) and viewed through a fluorescence microscope Olympus IX71 (Olympus, Tokyo, Japan).

Hepatocellular carcinoma Huh7 cells were cultured in 10% FBS-DMEM (Gibco; Invitrogen, Carlsbad, CA, USA) containing 0.1 mM non-essential amino acid, 2 mM l-glutamine, 36 µg/ml penicillin G, and 60 µg/ml streptomycin at 37 °C with 5% CO₂ and a humidified atmosphere. Dengue virus serotypes (DENV-1 strain Hawaii, DENV-2 strain 16681, DENV-3 strain H87, and DENV-4 strain H241) were propagated in mosquito C6/36 cells. A mouse monoclonal antibody specific for DENV NS1 (clones NS1-3F) was produced in our laboratory^{76 (link)}. Alexa Fluor 488-conjugated goat anti-mouse IgG antibody and Cy3-conjugated goat anti-mouse IgG antibody were purchased from Invitrogen and Jackson ImmunoResearch (West Grove, PA, USA), respectively. Peptide 3 (QFGPVFTWLNHA), peptide 4 (SFVNLWTPRYSL), peptide 10 (WHWRLWDVPDNP), and peptide 11 (WHWAWYSPTARM) that contained additional N-terminal 5-carboxyfluorescein (5-FAM) and C-terminal cell-penetrating peptide tag (RRRGRRRRRRRR) were synthesized at GenScript (Piscataway, NJ, USA) with purity of 97.9%, 96.4%, 98.5%, and 96.9%, respectively, and removal of standard trifluoroacetic acid.

The human monocytic cell line (purchased from CTCC, Wuhan University, Wuhan, China) was cultured as previously described (Xie et al., 2017 (link)). psiRNA-hTLR2, psiRNA-hTLR4, psiRNA-hTLR6, pZERO-hTLR2, pZERO-hTLR4, pZERO-hTLR6, pDeNy-hMyD88, pDeNy-hMal, and control plasmids (psiRNA-LucGL3, pZERO-mcs, and pDeNy-mcs) were obtained from InvivoGen (Carlsbad, United States). The inhibitor of NF-κB, BAY11-7082, was obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-phosphorylated and anti-total IκBα and p65 mouse antibodies were obtained from Abcam (Cambridge, MA, United States). Anti-β-actin antibody was bought from Sigma-Aldrich (St, Louis, MO, United States). Cy3-conjugated goat anti-mouse IgG antibody was obtained from Invitrogen (Frederick, MD). Antibodies against TLR2, TLR4, TLR6, MyD88, and Mal were purchased from Abcam (Cambridge, MA, United States). Neutralizing antibodies against TLR2, TLR4, and TLR6 were obtained from Novus. Complete protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Other reagents were purchased from Thermo Fisher Scientific (Waltham, United States).