Assay was performed as described [17] Assay was performed as des

Assay was performed as described [17]. Assay was performed as described [39] with some modifications. Anti-Syk immunoprecipitates from pervanadate stimulated RBL-2H3 cells,

used as source of active enzyme, and anti-Hrs immunoprecipitates from unstimulated RBL cells, used as substrate, were washed five times with lysis buffer, once with the kinase buffer (30 mM Hepes, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, and 100 μM Na3VO4), mixed, and resuspended in 40 μL kinase buffer containing 10 μCi of (γ-32P) ATP and 1 μM cold ATP. After 10 min of incubation at 30°C, beads were washed three times with lysis buffer, eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. Sensitized RBL-2H3 cells (5 × 105) were resuspended in 50 μL of serum-free medium and stimulated with 1 μg/mL DNP-HSA

for 30 min at 37°C. Endocytosis was check details stopped by addition of 0.1% NaN3 in cold PBS for 5 min. Samples were labeled with FITC-conjugated anti-mouse IgE and the cytofluorimetric analysis was performed with a FACSCalibur flow PF 2341066 cytometer (Becton Dickinson Immunocytometry Systems). Cells (120 × 103/well) were grown on glass coverslips coated with 2% gelatin, incubated with anti-DNP IgE (0.3 μg/well) overnight and stimulated with 500 or 50 ng/mL DNP-HSA for the indicated lengths of time to induce receptor internalization. Cells were then fixed, permeabilized, and stained with FITC-conjugated anti-IgE, as previously described [11]. To identify late

endosomes and lysosomes, cells were incubated with 300 nM Lyso-Tracker Red for the last 30 min during stimulation. Adenosine triphosphate Images were acquired at room temperature using an ApoTome Observer Z.1 microscope (Carl Zeiss, Jena, Germany) with a Plan-Neofluar objective x40/0.75 and an Axiocam MRm camera (all from Carl Zeiss). ApoTome Zeiss system provides an optical slice view reconstructed from fluorescent samples using a series of “grid projection” acquisitions, as reported [11]. Imaging stacks in the axial direction were acquired using AxioVision 4.6.3 software (Carl Zeiss), and all images shown are from a representative axial plane. Colocalization of the fluorescence signal was analyzed with AxioVision 4.6.3 software (Carl Zeiss). Images were processed with Photoshop 7 (Adobe, San Jose, CA, USA). The bands from immunoblot were quantified by densitometric analysis performed using Image J statistical software (National Institutes of Health, Bethesda, MD, USA). Data are presented as mean ± SD and compared using one-way analysis of variance followed by Student’s t-test. A p-value less than 0.05 was considered as statistically significant. We thank G. Benigni for isolating mouse bone marrow cells, G. Bernardini and A. Kettner for technical advises for BMMC culture, P. Birarelli and B. Milana for technical assistance, and P. Di Russo for secretarial assistance.

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