142 The Journal of Cell Biology | Volume 155, Number 1, 2001 to proceed for 4 h. To quantify adherent bacteria, the infected cell layers were extensively washed, resuspended by scraping into GCB liquid me- dium, serially diluted, and spread onto GCB plates. Bacteria were grown overnight and colony-forming units were counted. To quantify internalized bacteria, the infected cell layers were extensively washed, incubated with 150 g/ml gentamicin at 37 C for 1 h, and washed extensively before scraping and plating dilutions onto GCB plates. Each condition was per- formed in triplicate and each experiment was carried out four times inde- pendently. Immunoprecipitations and immunoblotting Cells were washed once with cold PBS and lysed in a NP-40–based buffer (0.1% NP-40, 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM EDTA, 2 mM NaVO4, 5 mM NaF, 1 mM PMSF, 10 g/ml each of aprotinin/leupep- tin/pepstatin). For the coimmunoprecipitation assay, cells were lysed in a Triton X-100–containing buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM MgCl2, 1 mM CaCl2, 2 mM NaVO4, 5 mM NaF, 1 mM PMSF, 10 g/ml each of aprotinin/leupeptin/ pepstatin). The insoluble fraction was removed by centrifugation and the cleared lysates were used for immunoprecipitation with specific antibodies or pull down experiment with the GST fusion protein. Precipitated proteins or fractions of the whole cell lysate were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose (Schleicher & Schuell). After blocking for 1 h in PBS/1% BSA/0.05% Tween 20, filters were probed overnight with specific antibodies. Proteins were visualized with peroxidase-coupled secondary antibody using the ECL system (Amersham Pharmacia Biotech). Src autophosphorylation/kinase assays Immunoprecipitation of src was carried out as described in the previous section, using anti-p60c-src antibody and protein G–Sepharose beads. Beads were washed three times in an autophosphorylation buffer (20 mM Pipes, pH 7, 10 mM MnCl2, 1 mM PMSF, 0.1 mM NaVO4, 2 g/ml aprotinin/leu- peptin/pepstatin). Beads were resuspended in autophosphorylation buffer and incubated for 5 min at 30 C with 1 M ATP and 25 Ci 32P ATP. Alter- natively, beads were washed three times in an enolase buffer (100 mM Pipes, pH 7, 10 mM MnCl2, 1 mM PMSF, 0.1 mM NaVO4, 2 g/ml aproti- nin/leupeptin/pepstatin) and incubated for 5 min at 30 C with 3 M ATP, 25 Ci 32P ATP, and 5 g of denatured enolase (5 min at 30 C in 25 mM acetate buffer, pH 3.3). Reactions were stopped with Laemmli’s buffer. Samples were heated for 10 min at 100 C, separated by electrophoresis on a 12% SDS-polyacrylamide gel, and transferred to nitrocellulose mem- branes. Incorporations of radioactive ATP by src ro enolase were detected by autoradiography. Confocal immunofluorescence microscopy HBMECs were grown to confluence on Permanox chamber slides (Lab- Tek). After infection, cells were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were blocked for 30 min with 3% BSA in PBS and were incubated for 2 h with the primary antibodies. After three washes with PBS, cells were incubated for 1 h with CY3-conjugated anti–mouse IgG and CY2-conjugated anti–rabbit IgG (Jackson ImmunoResearch Labo- ratories). Actin staining was performed with rhodamine-labeled phalloidin (Sigma-Aldrich) and bacteria staining with 0.5 g/ml ethidium bromide. Labeled preparations were mounted in mowiol and analyzed with a confo- cal microscope (Bio-Rad Laboratories). We thank Dr. B.B. 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