|
|
Blood, Vol. 104, Issue 4, 1100-1109, August 15, 2004
Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of I B kinase activity
Blood Bhattacharyya et al.
104: 1100
Supplemental materials for: Bhattacharyya et al, Vol 104, Issue 4, 1100-1109
Files in this Data Supplement:
- Figure 1. IL-10 pretreatment of DCs inhibits phosphorylation of IKK1 and IKK2. (JPG, 43 KB)
-
Bone marrow–derived DCs were prepared from NOD, NOR, and BALB/c mice. DCs were pretreated with 50 ng/mL of IL-10 or left untreated for 24 hours and stimulated with 500 ng/mL of LPS for 30 minutes. Cytoplasmic extracts were prepared and the expression of phosphorylated IKK analyzed using specific anti–phospho-IKK1 (Ser180) or -IKK2 (Ser181) Ab. The same membrane was reprobed with anti-IKK1 and anti-IKK2 Abs. Data are representative of at least 3 independent experiments.
- Figure 2. Costimulation with IL-10 and LPS has no effect on I
 B protein degradation. (JPG, 50 KB)
-
DCs were costimulated with IL-10 (50 ng/mL) and LPS (500 ng/mL) or treated with LPS alone for the indicated times (in hours). Cytoplasmic extracts were analyzed for expression of IB , IB , IB , or -actin. Densitometric analyses represent the ratio of intensity of IB protein to -actin expression per unit area.
- Figure 3. Cell viability study (JPG, 41 KB)
-
7-amino-actinomycin D (7-AAD; BD PharMingen, San Diego, CA) staining was used to access the viability of DCs. DCs were pretreated with 50 ng/mL of IL-10 or left untreated for 24 hours and stimulated with 500 ng/mL of LPS or 10 µg/mL of anti-CD40 Ab for 24 hours. DCs were stained with 7-AAD as per the manufacturer’s instructions. Briefly, DCs were washed twice in cold PBS and then resuspended in 1× Binding Buffer (BD PharMingen) at a concentration of 1×106 cells/mL. 7-AAD staining solution was added to cells, mixed gently, and incubated for 15 minutes in the dark. Samples were analyzed by flow cytometry. DC staining with 7-AAD was considered to be nonviable. The control panel represents DCs without treatment. The data are representative of results generally obtained for DCs following the various treatments detailed in the study.
|
|
