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Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: Primary structure of the HIV-1 Gag polyprotein proline-rich loop that binds cyclophilin A and confers cyclophilin A dependence on HIV-1 replication. The numbers refer to amino acid residues with respect to the amino terminus of the Gag polyprotein. The asterisks indicate the positions of G221, P222, and A224, residues important to this study.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

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Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 A224E mutant virus is cyclosporine resistant in Jurkat cells and cyclosporine dependent in H9 cells. Infection of the Jurkat T-cell line (A) or of the H9 T-cell line (B) was initiated by transfection of proviral DNA pNL4-3 encoding the A224E amino acid mutation. Cyclosporine (CsA) was maintained in the cell culture medium at the indicated concentrations. The accumulation of extracellular virions in the culture supernatant resulting from viral spread through the culture is indicated by the reverse transcriptase (RT) activity in the supernatant at the indicated times posttransfection.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Infection, Transfection, Cell Culture, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: P222A, a Gag mutation that confers reduced cyclophilin A affinity to the Gag polyprotein, rescues the replication of HIV-1 mutant A224E in H9 cells when the two mutations are expressed in cis. Jurkat T cells (A) or H9 T cells (B) were transfected with the wild-type proviral DNA pNL4-3 or with pNL4-3 encoding the P222A/A224E double mutation. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Transfection

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: H9 cells express more cyclophilin A (CyPA) than do Jurkat cells. (A) Jurkat cells (lanes 1, 3, and 5) and H9 cells (lanes 2, 4, and 6 to 9) were grown to a density of 106/ml, lysed in radioimmunoprecipitation assay buffer, and processed for Western blotting with a primary anti-cyclophilin A antibody. Lanes 1 to 4, samples normalized by cell number. The quantity of soluble cell lysate loaded per lane, as measured by number of cell equivalents, is indicated above the lanes. Lanes 5 to 9, samples normalized by quantity of total protein in the soluble cell lysate. The numbers above the lanes indicate the amount of total protein loaded per well. (B) Replication of wild-type HIV-1 in Jurkat or H9 cells after initiation of infection with a common stock of virus produced by transfection of 293T cells with pNL4-3. RT, reverse transcriptase. (C) Virions were purified from the supernatants of the cultures used for panel B on day 8 postinfection. Virion-associated protein was probed in a Western blot with a primary anti-cyclophilin A antibody. Lanes 1 and 2, samples from Jurkat and H9 cell cultures, respectively, normalized for reverse transcriptase activity and capsid protein. Lanes 3 and 4, serial twofold dilutions of the H9 sample, as indicated.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Radio Immunoprecipitation, Western Blot, Infection, Produced, Transfection, Purification, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 proviruses expressing Gag mutants with attenuated cyclophilin A affinity cannot promote a spreading infection in Jurkat cells (A) but do replicate in H9 cells (B). Infection was initiated by direct transfection of wild-type or mutant proviral DNA pNL4-3 into the T-cell lines. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Expressing, Infection, Transfection, Mutagenesis

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 replication in H9 cells is resistant to inhibition by cyclosporine. Infection of Jurkat T cells (A) or of H9 T cells (B) was initiated by transfection of wild-type (WT) proviral DNA pNL4-3. Cyclosporine (CsA) was maintained in the cell culture medium for the duration of the experiment at the indicated concentrations. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Inhibition, Infection, Transfection, Cell Culture

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 Gag mutants expressed in cis do not rescue the replication of Vif-defective HIV-1 in H9 cells. Infection of H9 T cells was initiated by transfection of wild-type (WT) or mutant proviral DNA pNL4-3. RT, reverse transcriptase. The Vif− virus is pNL4-3 with a deletion in the vif open reading frame (21).

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Infection, Transfection, Mutagenesis

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence