Figure 3.

HIV alters the levels and distribution of Cx43 in HIV-infected astrocytes cultures but does not alter their total protein amount or phosphorylated isoforms. A, Immunofluorescence of uninfected and HIV-infected astrocytes cultures and confocal microscopy to identify nuclei (EtBr-2, red), p24 (blue, Alexa350), and Cx43 (green, FITC) demonstrate that HIV-positive astrocytes (p24-positive cells) have higher membrane and intracellular Cx43 staining than uninfected cells. Arrows denote HIV-infected astrocytes and the corresponding high Cx43 staining. Uninfected cells, negative for p24 staining, located around the HIV-infected astrocyte clusters, express typical levels of Cx43. The lower panels of immunofluorescence are enlarged areas of uninfected and HIV-infected astrocytes (see corresponding dotted boxes in p24 and Cx43 staining pictures) and the corresponding Cx43 staining. Scale bar, 160 μm. B, Western blot analysis for Cx43 and histone-1 was performed on lysates obtained from cultures of uninfected (C, control cells) and astrocytes infected with HIV_92UG021, HIV_JR-CSF, or HIV_ADA for 7, 14, 21, and 28 d. Histone-1 was used as loading control. No significant differences in amount and phosphorylation (NP, not phosphorylated; P2, isoforms phosphorylated-2) of Cx43 were observed with HIV infection, perhaps because of the low number of HIV-infected cells, 4.7 ± 2.8%, with respect to the numbers of uninfected cells. “S” corresponds to a standard lysate of human astrocytes used as positive control (n = 15).