Figure 3
Figure 3. Inhibition of Pyk2 activation attenuates M-tropic gp120–induced iDC migration. (A) iDCs were pretreated with vehicle (dimethyl sulfoxide) or tyrphostin A9 (5 μM) for 1 hour at 37°C. The migration of these cells in response to M-tropic gp120 (10 nM) (left panel) or AT-2–inactivated virions (3 μg/mL p24) was determined after 3 hours of incubation. *P < .05 versus the vehicle control. (B) iDCs infected with rAAV5-expressing GFP protein were analyzed for GFP expression 48 hours after infection (top left panel) using a Zeiss Axiovert 40 CFL microscope (40×/0.50 NA). The fluorescence micrographs are shown on the right, whereas the corresponding phase-contrast image is shown on the left of the panel. iDCs were then transduced with recombinant AAV5-expressing Pyk2 mutant (Pyk2MT) or Pyk2 wild-type (Pyk2WT). Overexpression of the mutant and wild-type Pyk2 was demonstrated by Western blot analysis with anti-Pyk2 antibodies 48 hours after transduction. (Top and bottom right panels) Anti–glyceraldehyde phosphate dehydrogenase antibody was used as an internal control. The bar graph at the bottom of the panels shows the quantitative analysis of Pyk2 expression obtained by densitometry. *P < .05 versus the vector control. Data represent mean ± SD of 3 independent experiments. The transduced cells were tested for their ability to migrate in response to M-tropic gp120 (10 nM) using the chemotaxis assay (bottom left panel). *P < .05 versus the M-tropic gp120–treated vector control. Data represent mean ± SD of 3 independent experiments. (C) iDCs were transfected with Pyk2-specific siRNA and nontargeting siRNA (NT siRNA) using nucleofection (Amaxa Biosystems). The knockdown of Pyk2 expression was analyzed by Western blot analysis with anti-Pyk2 antibodies (left panel). Antiactin antibody served as a control. The bar graph at the bottom of the panel shows the quantitative analysis of Pyk2 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. Data represent mean ± SD of 3 independent experiments.

Inhibition of Pyk2 activation attenuates M-tropic gp120–induced iDC migration. (A) iDCs were pretreated with vehicle (dimethyl sulfoxide) or tyrphostin A9 (5 μM) for 1 hour at 37°C. The migration of these cells in response to M-tropic gp120 (10 nM) (left panel) or AT-2–inactivated virions (3 μg/mL p24) was determined after 3 hours of incubation. *P < .05 versus the vehicle control. (B) iDCs infected with rAAV5-expressing GFP protein were analyzed for GFP expression 48 hours after infection (top left panel) using a Zeiss Axiovert 40 CFL microscope (40×/0.50 NA). The fluorescence micrographs are shown on the right, whereas the corresponding phase-contrast image is shown on the left of the panel. iDCs were then transduced with recombinant AAV5-expressing Pyk2 mutant (Pyk2MT) or Pyk2 wild-type (Pyk2WT). Overexpression of the mutant and wild-type Pyk2 was demonstrated by Western blot analysis with anti-Pyk2 antibodies 48 hours after transduction. (Top and bottom right panels) Anti–glyceraldehyde phosphate dehydrogenase antibody was used as an internal control. The bar graph at the bottom of the panels shows the quantitative analysis of Pyk2 expression obtained by densitometry. *P < .05 versus the vector control. Data represent mean ± SD of 3 independent experiments. The transduced cells were tested for their ability to migrate in response to M-tropic gp120 (10 nM) using the chemotaxis assay (bottom left panel). *P < .05 versus the M-tropic gp120–treated vector control. Data represent mean ± SD of 3 independent experiments. (C) iDCs were transfected with Pyk2-specific siRNA and nontargeting siRNA (NT siRNA) using nucleofection (Amaxa Biosystems). The knockdown of Pyk2 expression was analyzed by Western blot analysis with anti-Pyk2 antibodies (left panel). Antiactin antibody served as a control. The bar graph at the bottom of the panel shows the quantitative analysis of Pyk2 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. Data represent mean ± SD of 3 independent experiments.

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