Nd antibodiesConclusion Our findings have important implications given the increased use
Nd antibodiesConclusion Our findings have important implications given the increased use of integrase inhibitors in antiretroviral PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 therapy. In the presence of integrase inhibitors uDNA accumulates because more substrates are available forTY191F [69], rodent TASK-1, rodent TASK-3 and IRK-1 expression plasmids were kind gifts from Dr. Douglas Bayliss (University of Virginia Charlottesville, VA). Human TASK-1, human TASK-3 and RelA-myc expression vectors were obtained from Origene (Rockville, MD). The Vpu-eGFP expression vector was a kind gift from Dr. Vasundhara Varthakavi (National Institutes of Health Bethesda, MD). Expression plasmids for codon-optimized Vpu (pcDNA-Vphu), phosphorylation defective mutant pcDNA-Vphu2/6 and a polyclonal antibody to Vpu were generous gifts from Klaus Strebel (National Institutes of Health, Bethesda, MD) and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25609842 have been described elsewhere [32]. A monoclonal antibody against HIV-1 Gag (GagM1) was generated in our laboratory. Commercially available antibodies against HIV-1 Gag KC57-RD1 (Beckman Coulter), TASK-1 (Chemicon) and -tubulin (Santa Cruz Biotechnology) were used for immunoblotting experiments. Secondary antibodies (horseradish peroxidase-Emeagwali and Hildreth Virology Journal 2012, 9:277 http://www.virologyj.com/content/9/1/Page 10 ofconjugated goat anti-rabbit (HRP-GAM), goat anti-mouse H+L (GAMH+L) and Fc specific (GAMFc) were obtained from Jackson ImmunoResearch Laboratories. Cell viability was measured using the Invitrogen fluorescence based LIVE/DEAD cell assay kit.Virusesthe anti-p24 antibody or expressing CFP, YFP or GFP as PD0325901 web appropriate.Virus titer determinationsHIV-1 was prepared in 293T cells by transfection of plasmid DNA (pNL4-3-CFP and pNL4-3 D116N-YFP (kind gifts from Dr. David Levi) [26], and pNL4-3, pNL4-3-Udel (kind gift from Dr. Klaus Strebel)) using Lipofectamine (Invitrogen) per the manufacturer’s instructions. The supernatants containing virus particles were harvested after 48hr of culture, filtered through a 0.2 micron filter and pelleted through a 20 sucrose cushion (100,000 , 1hr). All virus preparations were treated with DNase I to remove plasmid DNA.CellsTwo days after transfection with virus DNA constructs, HIV-1 Gag was measured in the cell culture supernatants and cell lysates using a capture ELISA.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblottingThe Jurkat T cell line was obtained from the American Type Culture Collection (ATCC) and cultured in complete RPMI (cRPMI) (RPMI, 10 mM HEPES, 2 mM L-glutamine, 10 fetal bovine serum (FBS). The JLTRG cell line was obtained from Dr. Olaf Kutsch through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: JLTRG (Cat #11587). HEK 293T cell line was obtained from the ATCC and cultured in complete Dulbecco modified Eagle medium (cDMEM) DMEM, 10 mM HEPES, 2 mM L-glutamine, 10 fetal bovine serum (FBS). Cell viability was assessed by trypan blue exclusion.Infections and transfectionsCell lysates were prepared by adding RIPA lysis buffer containing protease inhibitor cocktail (Roche) to cell pellets followed by incubation for 30 min on ice. Protein concentrations of cell lysates were determined using a bicinchoninic acid protein assay kit (Pierce-Rockford, IL). Equal amounts of protein were taken up in gel loading buffer and the samples were heated at 70 for 10 min before loading onto a 4 – 12 sodium dodecyl sulfatepolyacrylamide gel electrophoresis gradient gels (NuPAGE Novex Bis-T.
