Journal: PLoS ONE

Article Title: Ca 2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

doi: 10.1371/journal.pone.0074248

Figure Lengend Snippet: Cells were transfected with anti-Cav-1 siRNA or with a non-effective scrambled vector (mock cells). Then, silenced or mock cells were incubated with 35 nM ACT at 37°C for 10 min and surface staining of ACT followed in a flow cytometer. Internalisation of ACT was determined by FACS as described in Materials and Methods ( A ). Western blotting detection of caveolin-1 protein expression in CHO-K1 cells after transfection with the siRNA molecules ( B ). Cells were transfected with anti-Clathrin siRNA or with a non-effective scrambled vector (mock cells). Then, silenced or mock cells were incubated with 35 nM ACT at 37°C for 10 min and surface staining of ACT followed in a flow cytometer. Internalisation of ACT was determined by FACS as described in Materials and Methods ( C ). Western blotting detection of clathrin expression in CHO-K1 cells after transfection with the siRNA molecules ( D ). The procedure was performed as described in Materials and Methods . Equal loading of protein was confirmed in each blot by membrane stripping and further incubation with antibodies to visualize cytosolic GAPDH protein (Lower panel of the figure). The extent of protein silencing was determined by quantitative densitometric analysis. Data shown in the left-hand panels of the figure are the mean ± SD of at least three independent experiments, with *p<0.001.

Article Snippet: Anti-RTX, clathrin HC siRNA, caveolin-1 siRNA and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-caveolin-1, Cytochalasin D, methyl-β-cyclodextrin, nocodazole, nystatin, filipin, nifedipine, chlorpromazine, DMA, and genistein from Sigma-Aldrich (St Louis, MI, USA); anti-CD11b, anti-GAPDH, anti-P-caveolin-1 and anti-clathrin were from abcam (Cambridge, UK); KT5620, PP2 and PP3 were from Calbiochem (Merck, Germany); anti-mouse Texas Red, anti-rabbit FITC and BODIPY® FL C5 lactosylceramide complexed to BSA, from Invitrogen, Molecular Probes (Carlsbad, CA, USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Staining, Flow Cytometry, Western Blot, Expressing, Stripping Membranes

Journal: PLoS ONE

Article Title: Ca 2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

doi: 10.1371/journal.pone.0074248

Figure Lengend Snippet: Phosphorylation of Cav-1 in Tyr 14 was detected by Western blot of cell lysates incubated with the toxin (35 nM) for 5 min at 37°C ( A ). Caveolin-1 phosphorylation is significantly raised in the ACT-treated cells compared to control cells. ACT treatment raised about three times the Cav-1 phosphorylation in target cells. In addition, incubation of cells with PP2 (5 nM) significantly decreased the degree of Cav-1 phosphorylation, being similar to control cells. Incubation with PP3, the negative control of PP2, did not affect the Cav-1 phosphorylation induced by ACT treatment. Equal loading of protein was confirmed using specific antibodies to cytosolic GAPDH protein, after membrane stripping. Intensity of the bands was determined by scanning densitometry. ( B ) Effect of PP2 and PP3 on the ACT internalisation extent. CHO-K1 cells were pre-incubated for 30 min at 37°C with PP2 (5 nM) or PP3 (2,7 µM). Then cells were incubated with 35 nM ACT at 37°C for 10 min. Then, the surface staining of ACT was measured using a flow cytometer. Internalisation was assessed by FACS and expressed as described in Materials and Methods . Data shown in the lower panel are the mean ± SD of at least three independent experiments, with **p<0.025.

Article Snippet: Anti-RTX, clathrin HC siRNA, caveolin-1 siRNA and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-caveolin-1, Cytochalasin D, methyl-β-cyclodextrin, nocodazole, nystatin, filipin, nifedipine, chlorpromazine, DMA, and genistein from Sigma-Aldrich (St Louis, MI, USA); anti-CD11b, anti-GAPDH, anti-P-caveolin-1 and anti-clathrin were from abcam (Cambridge, UK); KT5620, PP2 and PP3 were from Calbiochem (Merck, Germany); anti-mouse Texas Red, anti-rabbit FITC and BODIPY® FL C5 lactosylceramide complexed to BSA, from Invitrogen, Molecular Probes (Carlsbad, CA, USA).

Techniques: Western Blot, Incubation, Negative Control, Stripping Membranes, Staining, Flow Cytometry