Figure 5
Figure 5. Activation of the LSP1 downstream of Pyk2 and p38 MAP kinase mediates gp120-induced iDC chemotaxis. (A) iDCs were untreated (0) or stimulated with M-tropic gp120 (10 nM) for the indicated time points. The cells were lysed and analyzed by Western blotting with p-LSP1 (left panel) antibody. The blots were stripped and reprobed with anti-LSP1 antibody. The bar graph (right panel) represents the phosphorylation index as obtained by densitometry. *P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. (B) iDCs cultured in chambered slides were treated with M-tropic gp120 (10 nM; left panel) or AT-2 HIV-1 (right panel) for 30 minutes. The cells were then fixed, permeabilized, and treated with rabbit anti-LSP1antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and 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. (C) iDCs were transfected with LSP1-specific siRNA and nontargeting siRNA (NT siRNA) using nucleofection (Amaxa Biosystems). The knockdown of LSP1 expression was analyzed by Western blot analysis with anti-LSP1 antibodies (left panel). Antiactin antibody served as a control. The bar graph at the bottom of the panel shows the quantitative analysis of LSP1 expression obtained by densitometry. *P < .05 versus the nontargeting vector control. Data represent mean ± SD of 3 independent experiments. The siRNA-transfected iDCs were used in the transwell migration assay in response to gp120 (10 nM; right panel). *P < .05 versus the M-tropic gp120–treated nontargeting siRNA control. (D) iDCs were pretreated with vehicle or tyrphostin A9 (5 μM) (left panel) or p38 MAP kinase inhibitor, SB203580 (30 μM; right panel), for 1 hour at 37°C. The cells were then stimulated with gp120 (10 nM) for 30 minutes. The cells were lysed and analyzed by Western blotting with anti–phospho-LSP1 antibody. The same blots were probed with anti-LSP1 antibody. The bar graph at the bottom of the panels represents the phosphorylation index as obtained by densitometry. *P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. Data show one representative experiment of 3 independent experiments.

Activation of the LSP1 downstream of Pyk2 and p38 MAP kinase mediates gp120-induced iDC chemotaxis. (A) iDCs were untreated (0) or stimulated with M-tropic gp120 (10 nM) for the indicated time points. The cells were lysed and analyzed by Western blotting with p-LSP1 (left panel) antibody. The blots were stripped and reprobed with anti-LSP1 antibody. The bar graph (right panel) represents the phosphorylation index as obtained by densitometry. *P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. (B) iDCs cultured in chambered slides were treated with M-tropic gp120 (10 nM; left panel) or AT-2 HIV-1 (right panel) for 30 minutes. The cells were then fixed, permeabilized, and treated with rabbit anti-LSP1antibody. After washing, cells were probed with Alexa 568–tagged anti–rabbit IgG antibody and 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. (C) iDCs were transfected with LSP1-specific siRNA and nontargeting siRNA (NT siRNA) using nucleofection (Amaxa Biosystems). The knockdown of LSP1 expression was analyzed by Western blot analysis with anti-LSP1 antibodies (left panel). Antiactin antibody served as a control. The bar graph at the bottom of the panel shows the quantitative analysis of LSP1 expression obtained by densitometry. *P < .05 versus the nontargeting vector control. Data represent mean ± SD of 3 independent experiments. The siRNA-transfected iDCs were used in the transwell migration assay in response to gp120 (10 nM; right panel). *P < .05 versus the M-tropic gp120–treated nontargeting siRNA control. (D) iDCs were pretreated with vehicle or tyrphostin A9 (5 μM) (left panel) or p38 MAP kinase inhibitor, SB203580 (30 μM; right panel), for 1 hour at 37°C. The cells were then stimulated with gp120 (10 nM) for 30 minutes. The cells were lysed and analyzed by Western blotting with anti–phospho-LSP1 antibody. The same blots were probed with anti-LSP1 antibody. The bar graph at the bottom of the panels represents the phosphorylation index as obtained by densitometry. *P < .05 versus the unstimulated control. Data represent mean ± SD of 3 independent experiments. Data show one representative experiment of 3 independent experiments.

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