Figure 1
Figure 1. M-tropic HIV-1 envelope causes activation of Pyk2 in iDCs. (A) The surface expression of DC-SIGN, CD14, HLA-DR, CD1a, CD4, CCR5, and CXCR4 on immature dendritic cells was analyzed by flow cytometry. Monocyte-derived immature dendritic cells harvested on the sixth day of differentiation were stained with FITC-conjugated antibodies for CD14, CD1a, and CCR5; PE-conjugated antibodies for DC-SIGN and CXCR4; and APC-conjugated antibodies for HLA-DR and CD4. Cells were also stained with FITC-, PE-, or APC-conjugated isotype antibody controls. Filled peaks indicate antibody control and open peaks indicate receptor expression. (B) iDCs were stimulated with M-tropic gp120 (10 nM) for the indicated periods of time at 37°C. The lysates were analyzed by Western blot analysis using antibodies to phospho-Pyk2 (Tyr 402; first panel), phospho-Pyk2 (Tyr 580; second panel), and phospho-Pyk2 (Tyr 881; third panel). The same blot was then probed with total Pyk2 antibody. iDCs were also stimulated with various concentrations of M-tropic gp120 (0-100 nM) for 15 minutes at 37°C. The lysates were analyzed by Western blot analysis using antibodies specific to phospho-Pyk2 (Tyr 402; fourth panel). iDCs were stimulated with T-tropic gp120 (10 nM) for the indicated periods of time at 37°C and lysates analyzed by Western blot analysis using antibodies specific to phospho-Pyk2 (Tyr 402; fifth panel). The phosphorylation indices of the respective blots are shown at the right of the panels. For quantitative analysis of protein phosphorylation, the ratio of phosphorylation versus total protein in each lane was obtained by densitometry. The phosphorylation index was determined by calculating the value of this ratio in each lane and presenting the ratio as the fold increase over the control value (unstimulated sample; 0), which was designated as 1. *P < .05 versus the unstimulated control. Values on the right panel are mean ± SD of 3 independent experiments. Data on the left panel show one representative experiment of 3 independent experiments. (C) iDCs were stimulated with AT-2–inactivated HIV-1 (YU2; 3 μg/mL p24) for the indicated periods of time at 37°C. The lysates were analyzed by Western blot analysis using antibodies to phospho-Pyk2 (Tyr 402; left panel). The same blot was then probed with total Pyk2 antibody. The phosphorylation index of the blot is shown in the right panel. *P < .05 versus the unstimulated control. Values on the right panel are mean ± SD of 3 independent experiments. Data on left panel show 1 representative experiment of 3 independent experiments.

M-tropic HIV-1 envelope causes activation of Pyk2 in iDCs. (A) The surface expression of DC-SIGN, CD14, HLA-DR, CD1a, CD4, CCR5, and CXCR4 on immature dendritic cells was analyzed by flow cytometry. Monocyte-derived immature dendritic cells harvested on the sixth day of differentiation were stained with FITC-conjugated antibodies for CD14, CD1a, and CCR5; PE-conjugated antibodies for DC-SIGN and CXCR4; and APC-conjugated antibodies for HLA-DR and CD4. Cells were also stained with FITC-, PE-, or APC-conjugated isotype antibody controls. Filled peaks indicate antibody control and open peaks indicate receptor expression. (B) iDCs were stimulated with M-tropic gp120 (10 nM) for the indicated periods of time at 37°C. The lysates were analyzed by Western blot analysis using antibodies to phospho-Pyk2 (Tyr 402; first panel), phospho-Pyk2 (Tyr 580; second panel), and phospho-Pyk2 (Tyr 881; third panel). The same blot was then probed with total Pyk2 antibody. iDCs were also stimulated with various concentrations of M-tropic gp120 (0-100 nM) for 15 minutes at 37°C. The lysates were analyzed by Western blot analysis using antibodies specific to phospho-Pyk2 (Tyr 402; fourth panel). iDCs were stimulated with T-tropic gp120 (10 nM) for the indicated periods of time at 37°C and lysates analyzed by Western blot analysis using antibodies specific to phospho-Pyk2 (Tyr 402; fifth panel). The phosphorylation indices of the respective blots are shown at the right of the panels. For quantitative analysis of protein phosphorylation, the ratio of phosphorylation versus total protein in each lane was obtained by densitometry. The phosphorylation index was determined by calculating the value of this ratio in each lane and presenting the ratio as the fold increase over the control value (unstimulated sample; 0), which was designated as 1. *P < .05 versus the unstimulated control. Values on the right panel are mean ± SD of 3 independent experiments. Data on the left panel show one representative experiment of 3 independent experiments. (C) iDCs were stimulated with AT-2–inactivated HIV-1 (YU2; 3 μg/mL p24) for the indicated periods of time at 37°C. The lysates were analyzed by Western blot analysis using antibodies to phospho-Pyk2 (Tyr 402; left panel). The same blot was then probed with total Pyk2 antibody. The phosphorylation index of the blot is shown in the right panel. *P < .05 versus the unstimulated control. Values on the right panel are mean ± SD of 3 independent experiments. Data on left panel show 1 representative experiment of 3 independent experiments.

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