cea4013-sup-0001-FigureS1.docxWord document22K Figure S1. Use of penicillin-specific IgE to detect changes in cell surface IgE levels. The assay was initially developed by removing some endogenous IgE from basophils using a lactic acid protocol previously used in numerous studies [1, 2]. However, it was necessary to document the sensitivity of this assay to detect the potentially low levels of BPO-specific IgE that might result from sensitization of basophils without lactic acid removal. Panel A: purified basophils were sensitized without lactic acid treatment with two concentrations of BPO-specific IgE. The experiments were done with multiple replicates to determine the natural variation in background and sensitized cells. The cells were also analyzed for unoccupied receptor density using a monoclonal anti-FceRI receptor antibody that only detects unoccupied receptors (15A5, Hoffmann-LaRoche). This antibody has been previously calibrated against absolute receptor densities, thus allowing an absolute ‘per-cell’ detection limit to be estimated from the variation in the replicates and background. See reference [3] for a more complete description of the calibration procedure. To calibrate the signal that detects BPO-specific IgE, one aliquot of cells was sensitized with a high concentration of BPO-specific IgE and one at a lower concentration. The cells were labeled with either 15A5 (or appropriate isotype control Ab) or by the procedure for detecting BPO-specific IgE. By flow cytometry (and running with pre-established PMT settings and calibrating against a standard bead), the net difference in the BPO-specific signal between the two loading conditions and the net difference in the 15A5 signal was determined (as the BPO-signal increases, the 15A5 signal decreases). Since 15A5 has been calibrated against an absolute receptor measurement, the delta for BPO-specific signal and the delta for the 15A5 signal sets the equation; BPO = 15A5*(calibrated value for 15A5). The figure shows an example of the linearity of the assay and the coefficient of variation that suggests the lower of sensitivity would be ~50 molecules of BPO-specific IgE.
cea4013-sup-0002-FigureS2.docxWord document19K Figure S2. Detection of IgE-mediated down-regulation of cell surface IgE (± PP1) using the BPO probe method. Basophils were sensitized (without lactic acid removal) and cultured with 10 µm PP1 or the equivalent amount of DMSO vehicle (a 1/2000 dilution) overnight with and without stimulation with 0.5 µg/mL anti-IgE Ab (6061). Flow cytometry of the remaining cell surface BPO-specific IgE (which presumably also indicates the total cell surface IgE density) was performed using the described procedure. Panels A and B: Examples of the flow cytography for this type of experiment; panel A, the gray filled histogram represents labeling with the anti-BPO-IgG in the presence of BPO-EACA (acts as the unlabeled control), the blue-filled histogram is the non-stimulated cells after overnight culture and the open red tracing, the stimulated cells after overnight culture (in this case 60% loss of signal). In panel B, gray is the unlabeled control, blue is the non-stimulated cells after overnight culture in 10 µm PP1 and the open red tracing the stimulated cells after overnight culture in 10 µm PP1 (in this case, 10% loss). Panel C; average of four experiments similar to those in panels A&B. Relative expression of BPO-specific IgE is plotted. These results are consistent with previous methods and functional changes [3, 4].
cea4013-sup-0003-FigureS3.docxWord document55K Figure S3. Histamine release induced by anti-IgE Ab (6061) before (●) and after (□) treatment for 3 days with 10 ng/mL IL-3.
cea4013-sup-0004-FigureS4.docxWord document24K Figure S4. Example Western blots for the signaling species examined. For the four signaling species, pErk, pSHIP, p-cbl, and p-Src, a single gel was run and the nitrocellulose cut at the approximately 64 kD line. The blotting order was phospho-Src followed by phospho-Erk for the portion < 64 kD and phospho-Cbl followed by phospho-SHIP followed by p85a. The order was chosen to take advantage of differences in the strength of the signals detected and the selectivity of the antibodies, with the weaker antibodies analyzed first. We have previously established [5] that p85a is a good lane-loading control antibody because its expression is unmodified during a wide variety of experimental designs and because it is easy to detect as the last step in this multiplex Western blot design. To detect syk phosphorylation, we prefer the use of immunoadsorption (IP) because existing phospho-syk antibodies show backgrounds that are not observed with immunoadsorption and are considerably less selective. Therefore, the bottom portion of figure shows the Western blots for syk phosphorylation run using cells stimulated in parallel with the cells used in top portion. Two time points were examined to confirm the expectation that for anti-IgE antibody, the 20 min time point was sufficient. Previous studies of later signaling such as Erk phosphorylation may be transient, but usually the period between 5–20 min is similar enough to not complicate the interpretation of using a single time point for these measurements. However, it is possible that the concentration-dependence curves could be skewed in favor of lower concentrations (the EC50 appearing leftward) because lower concentrations were only reaching their maximum by 20 min while higher concentrations may have been subsiding from their maximum at 20 min. Given the qualitative nature of the question, with signaling observed at very low concentrations, the timing should not be critical. The phospho-Src measurement includes three bands, p53, p56 and p59 (approximately). The p53 and p56 bands match well with the two lyn splice variants while the p59 band is unassigned. It might be consistent with fyn. In previous studies, we observe that the p53 form of lyn is the band that most clearly increases its state of phosphorylation. The reasons for the differences from p56 are not understood. We have also previously shown in some detail that the band representing c-cbl phosphorylation is broad and shows generally as a smear at approximately 110 kD [6]. Densitometric measurements readily cover the region but it isn't as obvious to the eye when presenting small slices of the blot as shown in the figure.
cea4013-sup-0005-FigureS5.docxWord document211K Figure S5. Synopsis of previously published studies of the relative concentration-dependence of histamine release vs. signaling element activation in basophils stimulated with an anti-IgE Ab. The figure is a synthesis of various studies in separate publications [2, 7-9]. (○) phospho-Shc, (●) phospho-SHIP1, (■) phospho-c-cbl, (♦) phospho-Erk 1/2, (□) phospho-syk, and (■) histamine release. The gray histogram represents the relative time-average of the cytosolic calcium response kinetics for a 15 min period of observation.
cea4013-sup-0006-FigureS6.docxWord document15K Figure S6. Cytosolic calcium response in purified basophils after 3 days of culture with 10 ng/mL IL-3 and stimulation with either 0.5 µg/mL () or 0.006 µg/mL of anti-IgE Ab (6061). The 0.006 µg/mL concentration was chosen to represent stimulation that was well below concentration needed to observe a significant basophil response in untreated basophils but still high enough to generate a strong response in basophils treated for 3 days with IL-3. The time-lag for this low concentration is consistent with previous studies of basophil stimulation [10].
cea4013-sup-0007-FigureS7.docxWord document18K Figure S7. Kinetic differences between syk phosphorylation and secretion of IL-4. Panel A: Basophils were sensitized with BPO-specific IgE and stimulated with an optimal concentration of BPO(12)-HSA for the times shown. Samples of supernatant were analyzed by an IL-4 ELISA (data is shown without normalizing for cell number) or cell pellets lysed and syk phosphorylation analyzed by Western blotting (relative phosphorylation expressed in band densities, arbitrary units). The plot shows one representative experiment of three. Panel B; basophils were sensitized with BPO-specific IgE and stimulated with BPO(12)-HSA. At 45 min, monovalent penicillin (BPO-EACA) was added to dissociate aggregates. Cell lysates were analyzed for IL-4 mRNA by real-time PCR. Previous studies provide the primers used for qPCR and have shown that IL-4 mRNA decays with a half-life of ~1–2 h when actinomycin D is added [11], a value that is consistent with the decay rate observed in this experiment after BPO-EACA was added. Previously published studies also noted that IL-4 secretion stops nearly immediately after addition of BPO-EACA
cea4013-sup-0008-Supplementary-Figures-Legends.docxWord document361K 

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