1. Loss of syk kinase during IgE-mediated stimulation of human basophils
Donald MacGlashan, MD, PhD, Katsushi Miura, MD, PhD 
Journal of Allergy and Clinical Immunology 
Volume 114, Issue 6, Pages (December 2004)
DOI: /j.jaci
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

2. Fig 1
Syk loss after IgE-mediated stimulation of basophils. A, A representative experiment. Actin was used to verify (and normalize for) equivalent lane loading. aIgE (o), optimal concentration of anti-IgE (0.2 μg/mL); aIgE (so), suboptimal concentration (0.002 μg/mL). Optimal caused 28% ± 11% histamine release, whereas suboptimal did not induce any measurable histamine release (as determined from the supernatants of these experiments). B, Distribution of syk loss during a 60-minute incubation period with 0.2 μg/mL anti-IgE antibody. C, Subset of experiments in which 18-hour incubations were also performed (n=3). D, E, similar experiments (n=3) using gp120-ovalbumin (D) or anti-IgE antibody (E). Optimal antigen in these experiments was 2 μg/mL (22% histamine release) and suboptimal, μg/mL (no measurable release), and the anti-IgE antibody concentration was 0.2 μg/mL (19% histamine release).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

3. Fig 2
Effects of proteasome inhibition on syk ubiquitylation. A, Cells stimulated with anti-IgE antibody for various times ± lactacystin, (100 μM). The blot (A) followed the anti-syk blot (B) after stripping to remove anti-syk antibody. Exposure was optimized for the broad antiubiquitin reactive zone >150 kd. B, Anti-syk blot for this experiment (1 of 4 experiments).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

4. Fig 3
Studies of syk ubiquitylation in basophils stimulated with anti-IgE antibody. A, An example of 1 experiment in which cells were stimulated without extracellular calcium (10 μM EDTA in PIPES-albumin-glucose) with anti-IgE at 0.2 μg/mL. The anti-syk (4D10) Western blot was overexposed to reveal the higher molecular weight species of 4D10+ bands. Molecular weight marker positions are shown to the left of the blot. B, The same blot after stripping and reblotting with antiubiquitin. The markers labeled B1 to B4 designate bands that increase in intensity after stimulation, and the 2 asterisks mark bands that are similar in unstimulated and stimulated cells. C, An anti-syk (4D10) blot of cell lysates (± anti-IgE for 60′) immunoprecipitated with antiubiquitin (1 of 2 similar experiments; 6 × 106 cells per condition). Because these bands are weak, a densitometric profile of the 2 right lanes is shown. D, An anti-syk (N-19) immunoblot of cell lysates (± anti-IgE for 60′) immunoprecipitated with anti-syk (4D10). E, Amount of anti-syk–positive material at Mr >p72 relative to the intensity of the p72 band.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

5. Fig 4
Characteristics of Cbl phosphorylation. A, Cbl phosphorylation after stimulation with 0.2 μg/mL anti-IgE antibody (1 of 4 experiments). After immunoprecipitation, blotting was performed first with antiphosphotyrosine (4G10), followed by stripping and reblotting with anti-syk (4D10). The 98-kD molecular weight marker is shown in the far left lane. The nitrocellulose was stripped again and blotted with anti-Cbl. B, Basophils stimulated under several conditions. The top blot shows stimulation with 0.2 μg/mL anti-IgE antibody ± extracellular calcium (1 of 4 experiments). The lower blot shows syk phosphorylation ± extracellular calcium (immunoprecipitation with anti-syk; 1 of 9 experiments). C, In the top blots, basophils were stimulated with 1 μM FMLP, 100 ng/mL PMA, 2 μM ionomycin, 10 ng/mL IL-3, or 0.2 μg/mL anti-IgE antibody. The FMLP-stimulated cells were lysed after 1 minute. Otherwise, cells were harvested after 10 minutes (1 of 2 experiments). The bottom blots show the effect of a 10-minute preincubation with buffer alone, 30 μM LY (a phosphatidylinositol 3′ kinase inhibitor; 1 of 5 experiments) or 10 μM PP1 (a lyn kinase inhibitor; 1 of 6 experiments). Cells were then stimulated for 5 minutes with anti-IgE antibody. IP, Immunoprecipitation.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

6. Fig 5
Kinetics of Cbl phosphorylation and syk loss. A, Phospho-Cbl during a 6-hour incubation after stimulation with 0.2 μg/mL anti-IgE antibody. B, The plot is the result of measuring the band intensities (○) of the Western blot, normalized for the presence of Cbl in each lane. The loss of syk (•) is shown (to calculate the loss, nonstimulated controls were included for the 5, 60, 240, 480, and 1080-minute time points). C, Concentration dependence of Cbl phosphorylation (•) after stimulation with anti-IgE antibody for 30 minutes (n=4). Included in the plot is the histamine release from the same samples (○) and, in 1 experiment, a parallel measurement of the loss of syk after an 18-hour incubation (□). In this 1 experiment, the relative phosphorylation of Cbl was 1.00, 0.92, 0.53, and 0.23 for 0.2, 0.02, 0.005, and μg/mL anti-IgE, respectively.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions