Ssrna41 lyovec

Two million freshly isolated PBMCs or 2 x 10⁵ monocytes incubated with or without 25,000 purified pDCs, were cultured in 1 ml of complete media and treated for 1–4 days with the TLR7/8 agonist R848 (Invivogen) at 1 μg/mL. Increasing concentrations of HIV-1 LTR derived GU-rich ssRNA40/LyoVec (5′GCCCGUCUGUUGUGUGACUC-3′) and negative control RNA, where U nucleotides were replaced with adenosine, ssRNA41/LyoVec (5′GCCCGACAGAAGAGAGACAC-3′) (26 (link)) (Invivogen), and recombinant human IFNα2A (PBL Interferon) were added to cells. The soluble IFN receptor, B18R (Affymetrix), was added (at 0.1μg/mL) to cells directly preceding stimulation with the above TLR7/8 agonists in order to neutralize type I IFN in the supernatant. For time course experiments, 500 μL of supernatant were removed and replaced with fresh media at 24 hr intervals. Concentrations of CXCL13 and IFNα in culture supernatants were determined by ELISA according to the manufacturers’ instructions (IFNα kit from PBL Biomedical Laboratories; CXCL13 kits from R & D systems).

Elacridar, Ko143, dipyridamole, and 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine (NBMPR) were purchased from Tocris (Minneapolis, MN). Verapamil was purchased from Sigma-Aldrich (St. Louis, MO). All compounds were dissolved in DMSO and stored at −20°C until use. CL097, ssRNA40/LyoVec and ssRNA41/LyoVec, and imiquimod were purchased from InvivoGen (San Diego, CA). The anti-MDR1/ABCB1 monoclonal antibody (D3H1Q) was obtained from Cell Signaling Technology (Danvers, MA). The anti-BCRP antibody (clone BXP-21) was obtained from Millipore Sigma (Burlington, MA). ENT1 (clone F-12) and ENT2 (clone A-8) monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). [³H]-Entecavir (6.4 Ci/mmol) and [adenine-2,8-³H]-Tenofovir (17.1 Ci/mmol) were acquired from Moravek Biochemicals, Inc. (Brea, CA). [N-methyl-3H]-Sofosbuvir (80 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Corning Gentest ABC transporter membranes were purchased from Corning (Corning, Woburn, MA).