CU-T12-9

CU-T12-9 specifically induces TLR1/2 activation, and facilitates the TLR1/2 heterodimeric complex formation, which in turn activates the downstream NF-κB signaling.^[1]

Cells were cultured in 200 μl of DMEM supplemented with 10% FBS, 10× penicillin/streptomycin, and 10× l-glutamine.  Cells were implanted in 96-well plates (4 × 10⁴ cells per well) for 24 hours at 37°C before drug treatment.  In the next 24 hours of treatment, medium was removed from the 96-well plate and replaced with 200 μl of supplemented Opti-MEM [0.5% FBS, penicillin (50 U/ml), streptomycin (50 μg/ml), 1× nonessential amino acids] containing 60 nM CU-T12-9, 0.66 nM (1 ng/ml) Pam3CSK4, or 0.77 nM (1 ng/ml) Pam2CSK4 according to the manufacturer’s protocol (InvivoGen), as well as different antibodies (0 to 10 μg/ml), including anti-hTLR1–IgG, anti-hTLR2–IgA, or anti-hTLR6–IgA. A sample buffer (20 μl) from each well of the cell culture supernatants was collected and transferred to a transparent 96-well plate.  Each well was treated with 200 μl of QUANTI-Blue buffer and incubated at 37°C for 1 h.