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Blood, Vol. 105, Issue 2, 742-749, January 15, 2005
Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo
Blood Kerksiek et al.
105: 742
Supplemental materials for: Kerksiek et al, Vol 105, Issue 2, 742-749
Files in this Data Supplement:
- Figure S1. Variability in the amount of activated Rac1 detected in GST pull-down assays performed on cultured DC from WT and Tg+ mice (PDF, 274 KB) -
Bone marrow-derived DC (1×107) were enriched for CD11c-positive cells and lysed as described in "Materials and Methods. " The lysate was centrifuged at 13,000 × g for 15 min at 4°C, and an aliquot of the supernatant was kept to assess the amount of protein in the total lysate. The supernatant was supplemented with 0.5% BSA and incubated with 50 mg GST-CRIB at 4°C for 15 min under rotation. GSH-sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden), washed in complete lysis buffer, were then added and the incubation continued for 1 h at 4°C with rotation. The beads were then washed 3 times at 4°C in lysis buffer and the samples analyzed by Western blot as described. Variability was encountered in analyses of both immature (as shown, 4 individual mice) and LPS-matured in vitro-cultured DC (n=7 WT and 8 Tg+). To our knowledge, GST pull-downs have never been performed under these conditions (i.e. low DN expression and multiple primary cell cultures); studies of DN Rho family function typically use transfection or microinjection to achieve high levels of DN protein in cell lines or single primary cell cultures, which may explain our inability to detect inhibition of Rac1 function with this method.
- Figure S2. Naive Tg+ mice have a normal distribution of DC in the spleen (PDF, 1652 KB) -
Serial sections from the spleen of a naive WT mouse were stained with CD11c-PE (red), Moma-biotin (+SA-Cy5, blue), and either B220-FITC (left, as in Fig. 4B) or Thy1.1-FITC (right). Because the localization of DC (red) in the T cell areas is easier to visualize when the T cells are unstained, we chose to preferentially use the antibody combination with B220-FITC.
- Figure S3. DC from CD11c-Rac(N17) mice show normal podosome formation and cell shape (PDF, 3394 KB) -
Immature (left) and LPS (1 µg/ml)-matured (right) in vitro-cultured DC from WT and Tg+ mice were plated onto fibronectin-coated coverslips, stained for CD11c (CD11c-biotin followed by SA-Cy5, blue), and then fixed with 4% paraformaldehyde/3% sucrose. Following permeabilization with 0.5% Triton, the cells were stained with the actin-specific toxin phalloidin (TRITC-conjugated, red) and purified anti-vinculin Ab. Goat antimouse Alexa 488 (green) was used to reveal the vinculin staining. The coverslips were mounted onto slides with Fluoromount G and analyzed by fluorescence microscopy as described for the spleen sections. Podosomes, migratory structures characterized by a ring of vinculin (green) containing points of actin (red), were detected in both WT and Tg+ immature DC (left). LPS-exposed DC from WT and Tg+ mice adopted the typical shape of mature DC, with extension of many actin-rich dendrites (right).
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