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Blood, Vol. 109, Issue 2, 653-660, January 15, 2007
Apoptotic cells induce Mer tyrosine kinase–dependent blockade of NF- B activation in dendritic cells
Blood Sen et al.
109: 653
Supplemental materials for: Sen et al, Vol 109, Issue 2, 653-660
Files in this Data Supplement:
- Figure S1. Inhibition of protein synthesis by Ebulin 1 blocks AC-mediated inhibition of NF-κB DNA binding activity (JPG, 61.1 KB)
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NOD BMDCs were preincubated with Eublin 1 (50 ng/mL) for 0.5 hours, treated with ACs for 3 hours, stimulated with LPS (50 ng/mL) for 0.5 hours, and nuclear NF- B DNA binding activity determined via EMSA.
- Figure S2. AC pretreatment blocks IκBα degradation and phosphorylation in BMDCs and sDCs (JPG, 58.3 KB)
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NOD BMDCs (A) or sDCs (B) were pretreated with ACs and stimulated with LPS as in Figure 1. (A) Expression of phospho (P)–IB in cytoplasmic extract was determined by Western blot. Densitometric analysis represents the ratio of intensity of phospho-IB to  -actin expression per unit area. (B) Cytoplasmic IB and -actin protein was detected by Western blot using the same blot. Densitometric readings represent the ratio of intensity of IB protein to -actin expression per unit area and are represented in arbitrary units.
- Figure S3. AC pretreatment inhibits NF-κB and IKK activation in B6 but not B6.MerTKKD BMDCs (JPG, 56.7 KB)
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B6 or B6.MerTKKD BMDCs were pretreated with ACs for 3 hours and then stimulated with LPS as in Figure 1. (A) DNA binding activity of nuclear NF-B or OCT-1 was determined via EMSA. (B) Phospho-IKK1 and -IKK2 were detected by Western blot, and the same blot was reprobed for IKK1 and IKK2 protein. Data are representative of 3 independent experiments.
- Figure S4. Pretreatment with PI3K inhibitors blocks AC-induced inhibition of NF-κB activation in BALB/c BMDCs (JPG, 58.9 KB)
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BALB/c BMDCs were preincubated with Wort (200 nM) or LY (50 µM) for 1 hour, treated with ACs for 3 hours or left untreated, and then stimulated with LPS (50 ng/mL) for 0.5 hours. (A) Nuclear NF-B or OCT-1 DNA binding activity was determined via EMSA. (B) Cytoplasmic IB protein was assessed via Western blot, and the same blot was reprobed for -actin protein.
- Figure S5. Pretreatment with ACs inhibits αCD40 Ab-induced NF-κB activation and IκBα degradation in NOD but not NOD.MerTKKD DCs (JPG, 76.8 KB)
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NOD and NOD.MerTKKD BMDCs were pretreated with ACs for indicated times or left untreated and then were stimulated with 10 µg/mL of hamster anti-CD40 (clone HM40-3) and 20 µg/mL antihamster (clone G188-2) Abs for 1 hour. (A) DNA binding activity of nuclear NF-B or OCT-1 was determined via EMSA. (B) Expression of cytoplasmic IB was analyzed by Western blot and the same blot reprobed for -actin. Corresponding densitometric analysis represents the ratio of intensity of IB expression to -actin expression per unit area.
- Figure S6. Axl and Tyro3 are not associated with immunoprecipitated MerTK (JPG, 35.6 KB)
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NOD BMDCs were pretreated with ACs and MerTK immunoprecipitated (IP) with MerTK Ab. Western blot was used to detect MerTK, Axl, and Tyro3 by reprobing the same membrane. Alternatively, whole-cell extracts prepared from AC-pretreated NOD BMDCs (WCE) or NOD thymocytes (THY; negative control) were similarly analyzed.
- Figure S7. AC-induced inhibition of NOD DC activation is mediated by PI3K signaling (JPG, 61 KB)
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NOD BMDCs were preincubated with 200 nM Wort or 50 µM LY and treated with ACs or left untreated. DC surface phenotype was determined via flow cytometry after 24 hours of culture, with or without LPS (100 ng/mL). Open and shaded histograms represent untreated versus LPS-treated NOD BMDCs, respectively.
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