Cytotoxicity experiments in HeLa37 cells were repeated at least three times wit

Resistant cells were sub sequently maintained in complete media supplemented with 0. 5 ug mL puromycin. pTRIPz viral stocks and stable cell lines were produced as described above for NFκB eGFP, except that the lentiviral vectors pTRIPz EV, pTRIPz DN IκB or pTRIPz p65 were used. Flow cytometry and staining Analysis of infected cells by flow cytometry and live MAPK 経路 cell sorting were performed as previously described. Of note, in all experiments, analysis was limited to live cells by FSC SSC gating at the time of data acquisition. Un less otherwise stated, infected cells were analyzed four days post infection. Jurkat E6 1 cells were stained and analyzed for CD69 as previously described except that antibodies were conjugated to PE Cy7 and 1 uL of antibody was used per 1 × 105 cells.

Jurkat cells were stained with PE Cy7 NFκB p65 and NFκB p50 with Pacific Blue conjugated secondary antibody as previously described. Compound treatments Infected cells were treated with the various compounds for the times and durations indicated in individual experiments. Compounds were added at the listed con centrations to complete media. Unless otherwise Linifanib 価格 stated, compounds were used at the following concentrations, TNF, 10 ng mL, SAHA, 0. 5 uM, PMA, 4 ng mL, Ionomycin, 1 uM, BMS 345541, 5 uM. Pyrosequencing of integration sites HIV 1 integration sites were analyzed by 454 deep sequencing as previously described. Briefly, Jurkat cells were infected with RGH and sorted into constituent populations three days post infection. Genomic DNA was extracted from 5 × 105 cells of each population, digested with MseI and ligated to adaptors.

Nested PCR with adapter and LTR specific primers was performed to amplify the HIV host genome junctions. Immunoblotting RGH infected Jurkat cells were lysed in NP 40 lysis buffer NP LY3009104 concentration 40, 0. 1% SDS supplemented with 1x protease inhibitor cocktail. Lysates were cleared by centrifugation, mixed with 4× SDS PAGE sample buffer, and boiled for 5 min. Whole cell extracts were separated on a 12% SDS PAGE gel and then transferred to nitrocellulose membrane. Membranes were blocked with 2% BSA in PBS Tween and then incubated with primary antibody overnight at 4 C. Antibodies used were as follows, IκB Abcam 32518, NFκB p65 Abcam 7970, GAPDH Abcam 9484. After washing and incubation with HRP conjugated secondary antibody, membranes were washed and signal was developed with SuperSignal West Femto chemi luminescent substrate.

Statistical analysis Unless otherwise stated, experiments were performed in biological triplicate. Where appropriate, statistical infer ence was performed on quantitative data. Two group testing was performed using the Students T test, while comparison between multiple groups was made using one way ANOVA followed by pairwise two group testing. Statistical analysis was per formed in R 2. 15. 1. Integration site analysis heatmaps were created using the INSIPID pipeline util izing previously described statistical methodology. Briefly, for each identified integration site, matched random controls were created in silico. This pairing of experimental and control sites allows for computation of relative enrichment and de enrichment profiles using a receiver operating characteristic framework.