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(A) Expression of Nef in the presence of wt, Änef, Äenv, Änef/Äenv (VSV-G) HIV-1 or mock infected immDCs.

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posted on 2013-03-16, 07:38 authored by Roni Sarkar, Debashis Mitra, Sekhar Chakrabarti

The immDCs were infected with the wild type and mutant viruses. After 24 hrs, the cells were harvested and assayed for Nef expression by immunoblotting. â-actin served as an internal control. 4. (B) Nef expressed by HIV-1 induces release of IL-6. The immDCs were infected with either wt, Änef, Äenv, Änef/Äenv (VSV-G) HIV-1. Cells were extensively washed and re-fed after infection. Supernatants were collected at 12 hrs and 24 hrs of infection. The content of IL-6 was then measured by ELISA. Data represent the mean±SEM (n = 3). Statistical analysis was performed using Student's t-test, with the levels of significance defined as p*<0.05 and p**<0.01. 4. (C) Flow cytometry analysis for the detection of infection in immDCs. After 24 hrs of infection with either wt, Δenv, Δnef or Δenv/Δnef were analysed for the detection of the HIV-1 antigen (p24) by incubating with anti-p24 HIV-1 monoclonal antibody followed by staining with PE-conjugated anti mouse antibody. PE-conjugated isotype matched IgG served as control. Presence of histogram peaks evidenced the occurrence of infection and validated our results.

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