Journal: Immunity

Article Title: Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation.

doi: 10.1016/j.immuni.2007.02.009

Figure Lengend Snippet: Figure 5. STAT5 Negatively Regulates Th17 Differentiation In Vitro and In Vivo (A) CD4+ T cells from Stat5a/bfl/flor Stat5a/bfl/fl; CD4-Cre mice were stimulated with anti-CD3 and anti-CD28 for 3 days in IL-2 alone or IL-2 together with either IL-23, anti-IFN-g, and anti-IL-4; or under optimal Th17 conditions, and cells were subsequently stained for IFN-g and IL-17. Representative data from two independent experiments are shown. (B) CD4+ T cells from Stat5a/bfl/flmice (white bars) or Stat5a/bfl/fl; CD4-Cre mice (black bars) were activated with anti-CD3 and anti-CD28, prior to measuring levels of IL-17 protein in culture supernatants by ELISA at 24, 48, and 72 hr. Histograms represent mean values and error bars represent SD values. The three asterisks denote a significant difference (p < 0.001) as determined by an unpaired t test. The values shown were measured in triplicate and are representative of two independent experiments. (C) Serum levels of IL-17 detected in wild-type (n = 5) and Stat5a/bfl/fl; CD4-Cre (n = 5) mice. The asterisk denotes a significant difference (p = 0.013) as determined by an unpaired t test.

Article Snippet: After preclearing with protein A agarose beads (Upstate, Charlottesville, VA), cell lysates from five million cells were immunoprecipitated with anti-STAT5 antibody (R&D Systems) or normal rabbit serum (Upstate USA, Chicago, IL) overnight at 4 C. After washing and elution, crosslinks were reversed at 65 C for 4 hr.

Techniques: In Vitro, In Vivo, Staining, Enzyme-linked Immunosorbent Assay

Journal: Immunity

Article Title: Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation.

doi: 10.1016/j.immuni.2007.02.009

Figure Lengend Snippet: Figure 6. Inhibition of Th17 Differentiation by IL-2 Requires STAT5 (A) CD4+ SP thymocytes from Stat5a/bfl/flor Stat5a/bfl/fl; CD4-Cre mice were stimulated with anti-CD3 and anti-CD28 for 3 days in media alone (Th0 conditions); together with IL-23, anti-IFN-g, and anti-IL-4; or under optimal Th17 conditions with and without the addition of 100 iu/ml IL-2. Cells were fixed and stained for IL-17 and IFN-g expression. The data shown are representative of three independent experiments. (B) Percentage of CD4+ T cells that expressed IL-17 was assessed by Student’s t test from three independent experiments with in vitro culture con- ditions as described in (A). Histograms represent mean values from Stat5a/bfl/fl(white bars) or Stat5a/bfl/fl; CD4-Cre (black bars) CD4+ SP thymocytes, and error bars represent SD values. Significantly different levels p < 0.05 and p < 0.01 are denoted with a single or double asterisk, respectively. (C) CD4+ T cells from C57Bl6 mice were polyclonally stimulated under neutral conditions for 3 days, rested in cytokine-free media (black bars), or stimulated with IL-2 (white bars). Cell lysates were immunoprecipitated either with anti-STAT 5 or normal rabbit serum (NRS). Bound DNA was an- alyzed by quantitative PCR with Il17a promoter site-specific primers to region D. Histograms represent mean values and error bars represent SD values. The double asterisk denotes significant difference (p < 0.01) as determined by an unpaired t test. The values were measured in triplicate and are representative of three independent experiments.

Article Snippet: After preclearing with protein A agarose beads (Upstate, Charlottesville, VA), cell lysates from five million cells were immunoprecipitated with anti-STAT5 antibody (R&D Systems) or normal rabbit serum (Upstate USA, Chicago, IL) overnight at 4 C. After washing and elution, crosslinks were reversed at 65 C for 4 hr.

Techniques: Inhibition, Staining, Expressing, In Vitro, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal of biomedical science

Article Title: Potent inhibition of HIV-1 replication by backbone cyclic peptides bearing the Rev arginine rich motif.

doi: 10.1007/s11373-007-9180-4

Figure Lengend Snippet: Figure 6 Inhibition by the Rev-BCPs of the Gag polyprotein precursor Pr55gag (upper panel), the Env glycoprotein precur- sor gp160env (lower panel) and the gp41env (middle panel) synthesis. Cellular extracts were obtained from HeLa MAGIC cells after one day of infection by HIV-1 Bru in the presence of Rev-BCPs (7 and 15) at different concentrations (25, 50 and 100 lM). After normalization by their total protein con- tent, extracts were analyzed by Western blot using a rabbit anti-p6gag or a rabbit anti-gp41env polyclonal serum and detected by chemiluminescence. Extracts from uninfected cells (first vertical lane), HIV-1 infected cells cultured in the absence of antiviral compounds (second lane) and HIV-1 infected cells cultured in the presence of AZT (10 lM), were used as controls

Article Snippet: Rabbit polyclonal anti-serum raised to gp41env was purchased from Fitzgerald Inc. and anti-p6gag was obtained after immunization of rabbits with GST-p6gag fusion protein.

Techniques: Inhibition, Infection, Western Blot, Cell Culture