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Blood, Vol. 110, Issue 13, 4341-4350, December 15, 2007
Immunoproteasome down-modulation enhances the ability of dendritic cells to stimulate antitumor immunity
Blood Dannull et al.
110: 4341
Supplemental materials for: Dannull et al
Files in this Data Supplement:
- Figure S1. Intracellular distribution of iPs are altered by iPsiRNA transfection of DC (PDF, 128 KB) -
DC derived from CD14+ monocytes were transfected with CsiRNA or iPsiRNA, then induced to mature using CC. Intracellular staining was then performed for LMP2, with the primary mAb being detected using a fluorescein-conjugated secondary antibody. Fluorescent micrographs are shown. Cells were visualized and images collected using an Olympus BX51 microscope, a 40× objective with an aperture of 0.75 and a final magnification of 100×, a Q-imaging camera (Retiga, Canada) and BioQuant acquisition software, with no additional image processing.
- Figure S2. Subunit Composition of Intact 20S Proteasomes in DC is Altered by iPsiRNA Transfection (PDF, 113 KB) -
(A) 2-D gel analysis of immunoprecipitated proteasomes was performed after metabolic labeling. Immature DC were transfected with either CsiRNA (top panel) or iPsiRNA (lowere, and after the induction of DC maturation using the CC were metabolically labeled. Immunoprecipitated proteasomes from cell lysates were then subjected to isoelectric focusing and then SDS PAGE. After transfer to nitrocellulose, proteasome subunits were visualized by autoradiography. Individual cP subunits X, Y, and Z are labeled accordingly. (B) Densitometry measurements for each cP subunit were normalized to the density of the  3 proteasome subunit on each gel and are depicted in bar graph format.
- Figure S3. The Phenotype of Mature DC is not Altered by siRNA Transfection (PDF, 149 KB) -
Monocytes derived iDC were left untransfected and not matured (iDC), were left untransfected and induced to mature using a CC (mDC), or were matured after transfection with control siRNA (mDC+CsiRNA) or with iPsiRNA (mDC+iPsiRNA). DC were then evaluated using 2-color flow cytometry for the indicated phenotypic markers.
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