|
|
Blood, Vol. 110, Issue 5, 1550-1558, September 1, 2007
Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function
Blood Kubach et al.
110: 1550
Supplemental materials for Kubach et al, Vol 110, Issue 5, 1550-1558
Files in this Data Supplement:
- Figure S1. Isolation and FACS analysis of CD25+ Tregs freshly isolated from leukapheresis products (JPG, 51.1 KB)
-
CD4+CD25− T cells and CD4+CD25+ Tregs were isolated from buffy coats and leukapheresis products (up to 1.5 × 1010 whole cells) of healthy volunteers.8;14 (A) Established protocols for isolation of CD4+CD25− T cells and CD25+ Tregs. (B) Freshly isolated CD25+ Tregs were stained with the indicated markers using fluorescence-labeled mAbs and analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Heidelberg, Germany). The figure shows the dot blot analysis of viable cells (dead cells excluded by PI staining).
- Figure S2. Galectin-10 expression in human CD25+ Tregs vs. human eosinophils (JPG, 42.9 KB)
-
Enlarged gel regions showing galectin-10 isoforms A-C in CD25+ Tregs compared to the corresponding region on 2D PAGE gels of human eosinophils. Gal-10 protein spots were identified by Western blot analysis and MALDI mass spectrometry.
- Figure S3. 2D PAGE pattern of soluble proteins of human CD25+ Tregs and galectin-10 expression in CD4+ T cells, CD25+α4β1 Tregs and CD25+α4β7 Tregs (JPG, 54.6 KB)
-
(A) Crude cell lysates were prepared and separated using the high-resolution 2D gel electrophoresis technique. Proteins with a pI range from 4 to 10 and a molecular weight from 5 kD to 150 kD are displayed. The sample load was 60 µg protein/10 µL sample and the proteins were silver stained. #:Due to gel size, the IEF rod gels were cut in two halves at approximately pI 6.0 to 6.4 before further processing. Arrowhead shows a cutting edge, which was due to technical reasons. In this context, please also see Figure S6. (B) Quantification of relative Gal-10 mRNA levels in freshly isolated CD4+ T cells, CD25+ 4 1 Tregs, and CD25+47 Tregs. cDNA samples were subjected to qRT-PCR. The relative quantities of Gal-10 mRNA were normalized according to the expression of -actin. (C) Silver stained 2D gel enlargements showing the different protein spot intensities of Gal-10 isoforms A-C in stimulated CD4+ T cells, CD25+41 Tregs, and CD25+47 Tregs.
- Figure S4. Galectin mRNA expression in CD4+CD25− T cells and CD25+ Tregs (JPG, 38.9 KB)
-
Galectin expression in CD25+ Tregs versus CD4+ T cells. Resting and stimulated CD25+ Tregs and CD4+ T cells were analyzed for the expression of the indicated galectins by RT-PCR.
- Figure S5. Analysis of siRNA-transfected human CD25+ Tregs (JPG, 63.7 KB)
-
On the left the siRNA transfection efficiency is shown, analyzed with fluorescence-labeled siRNA. The figure shows the flow cytometric analysis of CD25+ Tregs 48 hours after transfection with siRNA on the right. Depletion of dead cells using annexin-coupled beads (Miltenyi) resulted in a mean viability of cells greater than 92% as detected by propidium iodide exclusion.
- Figure S6. Procession of 2D PAGE of separated proteins of human T cells (JPG, 58.5 KB)
-
Crude cell lysates were prepared and separated using the high-resolution 2D gel electrophoresis technique. Proteins with a pI range from 4 to 10 and a molecular weight from 5 kD to 150 kD are displayed. Isoelectric focusing of each sample was carried out in rod gels of 40 cm in length. As there was no gel chamber available for gels of 30 × 40 cm, the IEF rod gel was cut in two halves at approximately pI 6.0 to 6.4 (as described in detail in Klose J. Large-gel 2-D electrophoresis. Methods Mol Biol. 1999;112:147-172.). Each IEF rod gel half was placed onto a 1D SDS gel of 25 × 30cm. Both SDS gels are run in parallel in the same gel chamber to ensure equal running conditions. Arrowheads show the cutting edges.
|
|
