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Blood, Vol. 113, Issue 7, 1526-1534, February 12, 2009
Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33
Blood Pecaric-Petkovic et al.
113: 1526
Supplemental materials for Pecaric-Petkovic et al
Sample preparation, gel electrophoresis and Western blot analysis
Cells (0.5–1 × 106 cells) were washed twice with ice-cold PBS, followed by the addition of 100% TCA to a final concentration of 10%. Proteins were allowed to precipitate on ice for 10 minutes, followed by a short sonication in order to prevent clumping of precipitated proteins. Protein precipitates were pelleted by centrifugation at 10,000 g for 15 min at 4°C. The supernatant was discarded and the protein precipitates were washed with 1 ml of cold aceton (−20°C). To ensure complete TCA removal, protein pellets resuspended in aceton were briefly sonicated until the suspension was homogeneous. Proteins were pelleted again by centrifugation. After an additional wash with acetone, pellets were taken directly in 1× reducing NuPAGE LDS Sample buffer (Invitrogen), and subjected to gel electrophoresis. To estimate protein concentration of the samples, an aliquot of the acetone suspension of each sample was pelleted separately and diluted in water. Protein concentration measurements were carried out using a commercial BCA Protein Assay (PIERCE). Proteins (15–20 µg per line) were separated in NuPAGE 12% Bis-Tris gels using MOPS SDS running buffer (Invitrogen) and electro-transferred onto polyvinylidene-difluoride membranes (Invitrolon, Invitrogen). Prestained molecular weight markers (Invitrogen) were included in each gel. Membranes were cut accordingly for the analysis of proteins of different molecular weight, blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20, and probed overnight with specific antibodies. Secondary horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies were from Bio-Rad (Bio-Rad, Switzerland; 1:3000 dilution). Immunoreactive bands were visualized using enhanced chemiluminsecence detection system (ECL-Kit, PIERCE) and Luminescent Image Analyzer (LAS3000, Fujifilm). Analysis of active, phosphorylated forms of proteins of similar molecular weight was always performed using non-stripped fresh duplicate membranes. To control the total amount of specific proteins, membranes were stripped by incubation in 0.2 N NaOH for 15–30 min at room temperature, blocked and reprobed with the antibodies which recognize total proteins. When indicated, for loading controls, stripped membranes were incubated with an anti–β-actin antibody. The quantitative analysis was performed using AIDA software (Fujifilm). Fold change of kinase activity (Erk’s and p38) was calculated as relative ratio of the band intensity value of phosphorylated form of the kinase in individual sample divided by the band intensity value of the phosphorylated kinase from control sample. The band intensity values of the phosphorylated form of the kinase were normalized to that of the total form. Western blot analysis was performed at least times using cells of different donors. Representative experiment are shown in the different figures. The following primary antibodies were used all at a 1:1000 dilution: phospho-Stat3 (Tyr705) rabbit mAb (D3A7), #9145; phospho-Stat5 (Tyr694) rabbit Ab, #9351; phospho-p44/42 MAPK (Thr202/Tyr204) rabbit mAb (20G11) #4376; p44/42 MAPK rabbit Ab, #9125; phospho-p38 MAPK (Thr180/Tyr182) rabbit Ab, #9211; p38 MAPK rabbit Ab, #9212; phospho–IκB-α (Ser32) rabbit Ab, #9241; IκB-α rabbit Ab, #9242; phospho–cPLA2 α (Ser505) rabbit Ab, #2831 (all from Cell Signaling); ST2 affinity purified goat Ab, AF523 (R&D); granzyme B mouse mAb (2C5), sc-8022 Santa-Cruz.
Files in this Data Supplement:
- Figure S1. Direct and synergistic induction of IL 4 and IL 13 secretion in response to IL 3 and IL 33 in basophils from different donors (JPG, 60.3 KB)
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Experimental conditions were as in Fig. 4A. Shown is the pair-wise analysis of different experimental conditions as indicated (ns, not significant, * p<0.05, ** p<0.01, *** p<0.001; paired t test). Each symbol represents the mean (of duplicates or triplicates) of a separate experiment and the data within the same experiment are connected by lines.
- Figure S2. IL-33 promotes cytokine secretion in basophils and eosinophils in a ST2-dependent manner (JPG, 42 KB)
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Basophils (A) or eosinophils (B) were preincubated for 15 minutes with a mixture of three anti-ST2 mAbs (a-ST2;clones 2A5, FB9, HB12; 3 µg/ml each) or IgG1 control mAb; (α-CD28 or irrelevant IgG1; 10 µg/ml) and then cultured for 24 hours with IL-3 or IL-3 + IL-33 (both at 10 ng/ml) as indicated in the figure. Shown are the mean values plus SD from three experiments. Note the almost complete block of the IL-33–mediated effect by the anti-ST2 antibodies in basophils and the prominent inhibition in eosinophils.
- Figure S3. Dose-response relationship of IL 33–induced IL 4 and IL 13 release (JPG, 49.3 KB)
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Basophils were incubated with anti-FcεRIα mAb 29C6 at a threshold (3 ng/ml) or maximally effective (100 ng/ml) concentration in the presence of increasing concentrations of IL 33 as indicated in the figure. IL 4 and IL 13 secretion was measured in the supernatants after 8 h of culture. Mean values of triplicates of a representative experiment out of three are shown.
- Figure S4. Preferential and dose-dependent activation of ERK and p38 in response to IL 3 and IL 33, respectively (JPG, 38.6 KB)
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Freshly isolated basophils were left untreated (nil) or stimulated with increasing concentrations of IL-3 or IL-33 for 10 minutes at 37°C as indicated in the figure and the activation of p38 and ERK was assessed by Western blotting. Actin is shown as loading control. A representative experiment is shown.
- Figure S5. Culture with IL 3 leading to ST2L induction does not strongly affect IL 33 signalling in blood basophils (JPG, 58.8 KB)
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Cells were cultured in medium or IL 3 (50 ng/ml) for 24 hours, washed and resuspended in fresh medium, followed by stimulation with IL 3 or IL 33 (at 50 ng/ml) for 5 and 15 min as indicated. The activation of Stat3/Stat5, the MAP-kinases Erk and p38, and the phosphorylation and degradation of IκB-α was analysed by Western blotting of the cell extracts. The 0 min time point shows the activation state of the cultured cells without stimulation. Also shown is the de novo induced sST2 and ST2L in the cells cultured with IL-3 but not in cells cultured in medium.
- Figure S6. In contrast to IL-3, short priming or prolonged preincubation with IL-33 does not enhance C5a-induced basophil degranulation (JPG, 25.6 KB)
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Basophils were preincubated for 10 minutes (Short priming) or cultured for 24 hours (Late priming) with either buffer control, IL-3 (10 ng/ml) or IL-33 (50 ng/ml) as indicated in the figure, followed by stimulation with C5a (10 nM) for 30 minutes when indicated by plus. The amount of histamine released into the supernatant is shown (mean plus SD of three experiments).
- Figure S7. Regulation of cytokine release by IL 1 family members in response to CD3/CD28-ligation in primary CRTH2+ Th2 cells and CCR5+ Th1 cells isolated from different donors (JPG, 48.2 KB)
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Experimental conditions were as in Fig. 6C. Shown is the pair-wise analysis of cytokine release in the presence or absence of the IL 1 family member (all at 50 ng/ml) indicated in the figure (ns not significant, * p<0.05; paired t test). The data from each separate experiment are connected by lines. Note the logarithmic scale for INF-γ secretion.
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