Figure 6
Figure 6. Gp120 induces LSP-1–actin association downstream of p38 MAP kinase. (A) iDCs were untreated (0) or treated with M-tropic gp120 and stained with Alexa Fluor 488–phalloidin (Invitrogen) and the cells were analyzed by flow cytometry. (B) iDCs were cultured in chambered slides, and either left untreated (C or 0) and were treated with M-tropic gp120 (top left panel) or AT-2–inactivated HIV-1 (bottom left panel) for 30 minutes. Then cells were fixed and treated with rabbit anti–LSP-1antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and Alexa Fluor 488–phalloidin. The slides were mounted using Prolong Gold antifade with DAPI (Invitrogen), and then examined under a Zeiss confocal microscope (63×/1.4 oil). The pictures were acquired using LSM 510 software. iDCs were unstimulated or stimulated with M-tropic gp120 (10 nM; top right panel) or AT-2 HIV (3 μg/mL p24; bottom right panel) for various time periods. The cells were lysed, and the lysates were immunoprecipitated with anti–LSP-1 antibody and immunoblotted with anti–β-actin antibody (top panel). The blots were stripped and reprobed with anti–LSP 1 antibody (bottom panel) to check the protein concentration in each lane. (C) iDCs were unstimulated or stimulated with M-tropic gp120 (10 nM) for 30 minutes in presence or absence of p38 inhibitor, SB203580 (30μM). The cells were lysed, and the lysates were immunoprecipitated with anti–LSP-1 antibody and Western blotted with anti–β-actin antibody (left panel). The blots were stripped and reprobed with anti–LSP-1 antibody to check the protein concentration in each lane. A vertical line has been inserted to indicate a repositioned gel lane. The bar graph at the bottom of the panel shows the quantitative analysis of LSP-1–actin association of the blots as obtained by densitometry.*P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. iDCs were cultured in chambered slides, then treated with M-tropic gp120 for 30 minutes in presence or absence of p38 inhibitor, SB203580 (30 μM). The cells were fixed and treated with rabbit anti–LSP-1 antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and Alexa Fluor 488–phalloidin (right panel). The slides were mounted using Prolong Gold antifade with DAPI (Invitrogen), and then examined under a Zeiss confocal microscope (63×/1.4 oil). The pictures were acquired using LSM 510 software.

Gp120 induces LSP-1–actin association downstream of p38 MAP kinase. (A) iDCs were untreated (0) or treated with M-tropic gp120 and stained with Alexa Fluor 488–phalloidin (Invitrogen) and the cells were analyzed by flow cytometry. (B) iDCs were cultured in chambered slides, and either left untreated (C or 0) and were treated with M-tropic gp120 (top left panel) or AT-2–inactivated HIV-1 (bottom left panel) for 30 minutes. Then cells were fixed and treated with rabbit anti–LSP-1antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and Alexa Fluor 488–phalloidin. The slides were mounted using Prolong Gold antifade with DAPI (Invitrogen), and then examined under a Zeiss confocal microscope (63×/1.4 oil). The pictures were acquired using LSM 510 software. iDCs were unstimulated or stimulated with M-tropic gp120 (10 nM; top right panel) or AT-2 HIV (3 μg/mL p24; bottom right panel) for various time periods. The cells were lysed, and the lysates were immunoprecipitated with anti–LSP-1 antibody and immunoblotted with anti–β-actin antibody (top panel). The blots were stripped and reprobed with anti–LSP 1 antibody (bottom panel) to check the protein concentration in each lane. (C) iDCs were unstimulated or stimulated with M-tropic gp120 (10 nM) for 30 minutes in presence or absence of p38 inhibitor, SB203580 (30μM). The cells were lysed, and the lysates were immunoprecipitated with anti–LSP-1 antibody and Western blotted with anti–β-actin antibody (left panel). The blots were stripped and reprobed with anti–LSP-1 antibody to check the protein concentration in each lane. A vertical line has been inserted to indicate a repositioned gel lane. The bar graph at the bottom of the panel shows the quantitative analysis of LSP-1–actin association of the blots as obtained by densitometry.*P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. iDCs were cultured in chambered slides, then treated with M-tropic gp120 for 30 minutes in presence or absence of p38 inhibitor, SB203580 (30 μM). The cells were fixed and treated with rabbit anti–LSP-1 antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and Alexa Fluor 488–phalloidin (right panel). The slides were mounted using Prolong Gold antifade with DAPI (Invitrogen), and then examined under a Zeiss confocal microscope (63×/1.4 oil). The pictures were acquired using LSM 510 software.

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