Figure 3
Figure 3. Tat induces SOCS-2 expression in CD14+ PBMos. The mRNA expression levels of SOCS-2 in response to Tat were verified by QRT-PCR using SOCS-2 TaqMan probe. (A) PBMos were untreated (UT), mock treated (M), or Tat treated at indicated concentrations for 4 hours. (B) PBMos were treated with 10 nM Tat at indicated time intervals. (C) The expression levels of SOCS-2 protein were confirmed by Western blotting using specific antibodies. PBMos were mock treated or Tat treated (10 nM) at indicated time intervals. After blotting to PVDF membrane, SOCS-2 protein was probed with specific anti–SOCS-2 antibodies. The SOCS-2 protein band was further verified by competitive Western blotting, of which cell lysates treated with Tat for 4 hours were probed with anti–SOCS-2 antibodies in the presence of purified recombinant SOCS-2 (rhSOCS-2, lane 7). Actin was used as a loading control. (D) HIV Tat expression in HIV-pseudovirus–treated macrophages. Primary blood macrophages were treated with control plasmid, HIV-pseudovirus, or Tat for 48 hours. Total cellular protein (70 μg) was collected and examined by Western blotting using anti-Tat antibodies and anti-STAT1 antibodies. Total STAT1 was probed as a loading control. A representative figure of independent experiments performed on macrophages from 3 different donors is shown. (E) Primary blood macrophages were treated with HIV-pseudovirus or with plasmids alone (mock) for 48 hours and total cellular protein was collected. The SOCS2 expression was measured by Western blot and probed against anti-SOCS2 and actin antibodies. Vertical lines have been inserted to indicate a repositioned gel lane. (F) PBMos were cycloheximide treated (CHX), or Tat treated (10 nM) in the absence or presence of CHX (40 μg/mL) at indicated time intervals. The expression levels of SOCS-2 mRNA were measured by QRT-PCR. ND refers to not detected. The statistical analysis was done by comparison between groups using the Student t test. *P < .05 was considered statistically significant. (G) The expression levels of TNFα and IL-10 from the cell-culture supernatants of PBMos, which were treated with Tat (10 nM) in the absence or presence of cycloheximide (40 μg/mL) at indicated time intervals, were analyzed using specific ELISA kit. All data presented were plotted as mean values ± SEM of at least 3 independent experiments in triplicates.

Tat induces SOCS-2 expression in CD14+ PBMos. The mRNA expression levels of SOCS-2 in response to Tat were verified by QRT-PCR using SOCS-2 TaqMan probe. (A) PBMos were untreated (UT), mock treated (M), or Tat treated at indicated concentrations for 4 hours. (B) PBMos were treated with 10 nM Tat at indicated time intervals. (C) The expression levels of SOCS-2 protein were confirmed by Western blotting using specific antibodies. PBMos were mock treated or Tat treated (10 nM) at indicated time intervals. After blotting to PVDF membrane, SOCS-2 protein was probed with specific anti–SOCS-2 antibodies. The SOCS-2 protein band was further verified by competitive Western blotting, of which cell lysates treated with Tat for 4 hours were probed with anti–SOCS-2 antibodies in the presence of purified recombinant SOCS-2 (rhSOCS-2, lane 7). Actin was used as a loading control. (D) HIV Tat expression in HIV-pseudovirus–treated macrophages. Primary blood macrophages were treated with control plasmid, HIV-pseudovirus, or Tat for 48 hours. Total cellular protein (70 μg) was collected and examined by Western blotting using anti-Tat antibodies and anti-STAT1 antibodies. Total STAT1 was probed as a loading control. A representative figure of independent experiments performed on macrophages from 3 different donors is shown. (E) Primary blood macrophages were treated with HIV-pseudovirus or with plasmids alone (mock) for 48 hours and total cellular protein was collected. The SOCS2 expression was measured by Western blot and probed against anti-SOCS2 and actin antibodies. Vertical lines have been inserted to indicate a repositioned gel lane. (F) PBMos were cycloheximide treated (CHX), or Tat treated (10 nM) in the absence or presence of CHX (40 μg/mL) at indicated time intervals. The expression levels of SOCS-2 mRNA were measured by QRT-PCR. ND refers to not detected. The statistical analysis was done by comparison between groups using the Student t test. *P < .05 was considered statistically significant. (G) The expression levels of TNFα and IL-10 from the cell-culture supernatants of PBMos, which were treated with Tat (10 nM) in the absence or presence of cycloheximide (40 μg/mL) at indicated time intervals, were analyzed using specific ELISA kit. All data presented were plotted as mean values ± SEM of at least 3 independent experiments in triplicates.

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