Journal: Journal of leukocyte biology

Article Title: HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro.

doi: 10.1189/jlb.0704424

Figure Lengend Snippet: Fig. 1. Up-regulation of IL-7R -chain by HIV-1 infection and Tat protein. (a) RT-PCR of IL-7R- RNA in uninfected (0) and HIV-1-infected () MDM. Cells were harvested on Day 10 postinfection. -Actin mRNA levels were measured to facilitate normalization. Serial dilutions of 1:2 (2)–1:16 (16), as indicated, were made of the RNA preparations after RT but before PCR. Results presented are typical of experiments on five different donors. Up-regulation of IL-7R RNA was not seen on Days 1 and 3 of culture (not shown). (b) RT-PCR of IL-7R- RNA in untreated (0) and Tat-treated () MDM. Tat protein was added to uninfected cells on Day 14 of culture. Cells were harvested 2 days later. Results are typical of experiments using five different donors. (c) Western blot analysis of IL-7R -chain. MDM were treated as in a and b before harvest. Results are typical of experiments on three different donors. Ab, Antibody. (d) Flow cytometry analysis of uninfected (Control) and HIV-1-infected MDM using mouse anti-human IL-7R and isotype control antiserum as indicated.

Article Snippet: The Western blot was probed with mouse anti-human IL-7R -chain antibody (R&D Systems, Minneapolis, MN) as the primary antibody and peroxidaseconjugated goat anti-mouse immunoglobulin G (IgG; Pierce, Rockford, IL) at a 1:15,000 dilution as the secondary antibody.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Control

Journal: Journal of leukocyte biology

Article Title: HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro.

doi: 10.1189/jlb.0704424

Figure Lengend Snippet: Fig. 2. Activation of STAT-3 by IL-7R signaling. Cells were left uninfected or infected with HIV for 10 days, or cells were left untreated or treated with Tat protein from Day 14 to Day 16 of culture before analysis of IL-7R signaling, where cells were rested in serum-free DMEM with gentamicin for 4 h at 37°C. During this rest period, no measurable IL-7 accumulated in the culture supernatants (less than 0.25 pg/ml by ELISA, data not shown). Cells were then harvested after 20 min of no treatment or 20 min of treatment with 100 ng/ml IL-7 for 20 min. Lysates run on Western blots were stained with rabbit antiserum specific for phosphorylated (activated) STAT-3 (anti-P-STAT-3). After exposure, blots to be “reprobed” were stripped and stained with antisera specific for total STAT-3. (a) HIV-infected MDM, probed with antisera as indicated. (b) Tat protein treatment, uninfected MDM, probed with antisera as indicated. Similar results were obtained in one additional experiment (using different donors than shown), in which the filters were stripped and reblotted with antisera to total STAT-3 as shown, and in two other additional experi- ments (using other, additional donors), in which the filters were not reprobed with antisera to total STAT-3. (b) Lanes 2 and 3 have been graphically switched (from their original gel positions).

Article Snippet: The Western blot was probed with mouse anti-human IL-7R -chain antibody (R&D Systems, Minneapolis, MN) as the primary antibody and peroxidaseconjugated goat anti-mouse immunoglobulin G (IgG; Pierce, Rockford, IL) at a 1:15,000 dilution as the secondary antibody.

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Staining