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Blood, Vol. 110, Issue 7, 2565-2568, October 1, 2007
TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells
Blood Nakae et al.
110: 2565
Supplemental materials for Nakae et al, Vol. 110, Issue 7, 2565-2568
Files in this Data Supplement:
- Document 1. Study design (PDF, 26.9 KB)
- Figure S1. Specificity of anti-TIM-1, anti-TIM-2 and anti-TIM-3 mAbs (JPG, 86.2 KB)
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The reactivity of anti-TIM-1 (RMT1-4 and RMT1-17), anti-TIM-2 (RMT2-1 and RMT2-14), and anti-TIM-3 (RMT3-23) monoclonal antibodies (mAbs) to mouse TIM transfectants was assessed by FACS. Nontransfected L5178Y cells (“-” in the figure), or L5178Y cells transfected with various TIM molecules, were stained with the indicated mAbs or control rat IgG followed by PE-conjugated antirat IgG. Bold lines show staining with indicated mAbs and dotted lines show background staining with control IgG.
- Figure S2. Specificity of anti-TIM-4 mAb (JPG, 35.8 KB)
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The specificity of the anti-TIM-4 (21H12) and anti-TIM1 (3B3) mAbs was assessed by ELISA. A plate was coated with mTIM-1-Ig, mTIM-3-Ig, or mTIM-4-Ig (2 mg/mL). The specificity of each mAb then was assessed by its ability to bind TIM-1-Ig, TIM-3-Ig or TIM-4-Ig. Binding was detected with antirat-kappa-HRP (Southern Biotech, Birmingham, ALA). Anti-TIM-4 mAb (21H12) bound TIM-4-Ig, but not TIM-1-Ig or TIM-3 Ig, whereas anti-TIM-1(3B3) bound TIM-1-Ig, but not TIM-3-Ig or TIM-4-Ig.
- Figure S3. Western blot analysis of TIM expression in mast cells (JPG, 41.1 KB)
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Whole cell lysates from naïve C57BL/6J BMCMCs (105 cells) were analyzed for expression of TIM-1 or TIM-3, using anti-TIM-1 or anti-TIM-3 mAbs or polyclonal antibodies (pAbs). The bands for TIM-1 and TIM-3 were at approximately 70kD to 80 kDa.
- Figure S4. Neither anti-TIM-3 pAb nor rmTIM-4 influence IgE/Ag-FcRI-mediated MAPK phosphorylation in BMCMCs (JPG, 54.5 KB)
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Expression of phosphorylated ERK1/2, JNK and p38MAPK in IgE/Ag-stimulated C57BL/6J-BMCMCs in the presence of 20 µg/mL anti-TIM-3 pAb or control goat IgG or in the presence or absence of 40 µg/mL rmTIM-4. In the figure, “0 Minutes” are baseline values in BMCMCs examined without antigen stimulation. Shaded areas are isotype-matched control IgG staining; solid lines are specific Ab staining. Green lines are BMCMCs cultured with goat IgG or no rmTIM-4 (medium), red lines are BMCMCs cultured with anti-TIM-3 pAb or rmTIM-4. Data show FACS results from one of the three experiments performed, using three distinct batches of C57BL/6J-BMCMCs, each of which gave similar results.
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