Pam3csk4 tlr1 2

Pam3CSK4 (TLR1/2) and LPS (TLR4) were obtained from InvivoGen (Carlsbad, CA, USA). Mouse recombinant IFN-β was purchased from PBL InterferonSource (now PBL Assay Science, Piscataway, NJ, USA) and mouse IFN-γ was from R&D Systems (Minneapolis, MN, USA). Antibodies used in this study were obtained as follows. Anti-H-PGDS and Anti-COX-2 antibodies were produced by Cayman Chemical (Ann Arbor, MI, USA). Anti-STAT1 and anti-pY701-STAT1 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-actin antibody was from BD Biosciences (Franklin Lakes, NJ, USA). PGs were purchased from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were from Sigma (St. Louis, MO, USA), unless stated otherwise.

The TLR agonists LPS (TLR4), R848 (TLR7/8), and Pam3CSK4 (TLR1/2) (all from Invivogen, San Diego, CA, USA) concentrations were previously optimized in TLR titration experiments. As no significant differences were observed in HIV infections (Supplementary Figure 1) or cytokine profiles (Supplementary Figures 2–4) between the TLR concentrations, a final concentration of 2 μg/ml was used. Phytohaemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO, USA), used as the positive control at a final concentration of 10μg/ml. Unstimulated PBMCs were used as the negative control. The CCR5-tropic HIV-1 NL4-3 AD8 (35 (link)) (a gift from Dr. Alex Sigal), was used at a working dilution of 1:20, which corresponded to an MOI of 0.9, which had been previously optimized (data not shown). PHA and unstimulated uninfected conditions were used as controls for HIV.