Figure 1.
HIV-1 gp120 and HIV-1 purification for attachment and tracking studies in MDDCs. (A) Binding specificity of biotin-LC-hydrazide gp120. (Ai) Specific binding of labeled gp120 to CLRs and CD4 was examined by preincubating MDDCs with either the CLR inhibitor mannan at 5 mg/mL, the CD4 mAb Leu3a at 10 μg/mL, or a combination of both prior to the addition of gp120. Flow cytometry of cells incubated under the following conditions are shown. (1) Media alone (filled gray panel), (2) Leu3a, (3) mannan, (4) Leu3a and mannan. Untreated cells were included for comparison (-ve). (Aii) Inhibition of HIV gp120 binding for each condition described for panel Ai was compared as percent gp120 binding. Mean fluorescence intensities (MFIs) were converted into percent binding by the following formulas: [(MFI of gp120 binding in the presence of inhibitors) – (MFI background {no gp120})] / [(MFI of the gp120 binding positive control {no inhibitors}) – (MFI background)]. (B) AT-2 HIV-1BaL purification and binding to MDDCs. (Bi) AT-2 HIV-1BaL was velocity-gradient purified as described in “Materials and methods,” and 5 μg HIV-1 p24 was lysed and diluted 1:2 in 1 × loading dye and subjected to SDS-PAGE and staining by Coomassie blue. Major protein bands p24 (capsid) and p17 (matrix) are indicated by arrows (left gel lane). Molecular weight markers are shown in the right lane. (Bii) Percent AT-2 HIV-1 and HIV-1 gp120 binding were compared in the presence of increasing concentrations of the CLR inhibitor, mannan. For detection of HIV-1 attachment, 1 × 105 cells/100 μL were preincubated with mannan for 1 hour and then 2 μg/mL p24 of purified virus at 4°C for 1 hour. Cells were washed, pelleted, and lysed and virus p24 was detected by ELISA. Percent HIV binding was calculated as follows: (sample – negative control)/(positive control). Negative control was media alone for HIV and gp120 binding. Positive controls were virions and gp120 binding in the absence of inhibitors for HIV and gp120, respectively. •, gp120; ○, HIV-1 virions. (Biii) Inhibition of AT-2 HIV-1 (▪; p24 ELISA) and gp120 (□; flow cytometry) binding to MDDCs was compared in the presence of (1) media alone, (2) Leu3a, (3) mannan, and (4) mannan and Leu3a as in panel Ai. All results shown are representative of at least 3 experiments. Error bars indicate standard deviation.

HIV-1 gp120 and HIV-1 purification for attachment and tracking studies in MDDCs. (A) Binding specificity of biotin-LC-hydrazide gp120. (Ai) Specific binding of labeled gp120 to CLRs and CD4 was examined by preincubating MDDCs with either the CLR inhibitor mannan at 5 mg/mL, the CD4 mAb Leu3a at 10 μg/mL, or a combination of both prior to the addition of gp120. Flow cytometry of cells incubated under the following conditions are shown. (1) Media alone (filled gray panel), (2) Leu3a, (3) mannan, (4) Leu3a and mannan. Untreated cells were included for comparison (-ve). (Aii) Inhibition of HIV gp120 binding for each condition described for panel Ai was compared as percent gp120 binding. Mean fluorescence intensities (MFIs) were converted into percent binding by the following formulas: [(MFI of gp120 binding in the presence of inhibitors) – (MFI background {no gp120})] / [(MFI of the gp120 binding positive control {no inhibitors}) – (MFI background)]. (B) AT-2 HIV-1BaL purification and binding to MDDCs. (Bi) AT-2 HIV-1BaL was velocity-gradient purified as described in “Materials and methods,” and 5 μg HIV-1 p24 was lysed and diluted 1:2 in 1 × loading dye and subjected to SDS-PAGE and staining by Coomassie blue. Major protein bands p24 (capsid) and p17 (matrix) are indicated by arrows (left gel lane). Molecular weight markers are shown in the right lane. (Bii) Percent AT-2 HIV-1 and HIV-1 gp120 binding were compared in the presence of increasing concentrations of the CLR inhibitor, mannan. For detection of HIV-1 attachment, 1 × 105 cells/100 μL were preincubated with mannan for 1 hour and then 2 μg/mL p24 of purified virus at 4°C for 1 hour. Cells were washed, pelleted, and lysed and virus p24 was detected by ELISA. Percent HIV binding was calculated as follows: (sample – negative control)/(positive control). Negative control was media alone for HIV and gp120 binding. Positive controls were virions and gp120 binding in the absence of inhibitors for HIV and gp120, respectively. •, gp120; ○, HIV-1 virions. (Biii) Inhibition of AT-2 HIV-1 (▪; p24 ELISA) and gp120 (□; flow cytometry) binding to MDDCs was compared in the presence of (1) media alone, (2) Leu3a, (3) mannan, and (4) mannan and Leu3a as in panel Ai. All results shown are representative of at least 3 experiments. Error bars indicate standard deviation.

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