Interferon-α Regulates Glutaminase 1 Promoter through STAT1 Phosphorylation: Relevance to HIV-1 Associated Neurocognitive Disorders
Figure 5
STAT1 binds directly with the GLS1 promoter in HIV-1 infected MDM.
(A-E). MDM were infected with or without HIV-1ADA for 5 days. (A). ChIP was performed using p-STAT1 (Tyr 701) antibody; IgG was used as a negative control. (B). Supernatants were tested for RTase activity. (C). p-STAT1 and STAT1 were detected by Western blot with β-actin used as a loading control. (D). Glutamate was detected in supernatants by HPLC. (E). Intracellular glutamate was detected using the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data (A-E) are representative of three independent experiments using three different donors. (F) Human MDM were infected with HIV-1 for 5 days, then total RNA was collected. IFN-α and IFN-β mRNA levels were determined by real-time RT-PCR. Results shown are representative of three independent experiments using three different donors and are means of triplicate samples. (G-I) Human MDM were infected with HIV-1 for 5 days in the presence of IgG or IFN-α/IFN-β neutralizing antibodies. Total RNA and cell lysates were collected and GAC mRNA (G) and protein (H and I) levels were determined by real time RT-PCR and Western blot, respectively. For quantification, GAC expression were normalized as a ratio to β-actin and shown as fold change relative to IgG control . Results (F, I) are shown as the average ± SEM of three independent experiments with three different donors, #, p<0.05, *, p<0.01, **, p<0.001, when compared with uninfected MDM or IgG control.