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Blood, Vol. 109, Issue 9, 3849-3855, May 1, 2007
Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myeloma
Blood Luciano et al.
109: 3849
Supplemental materials for: Luciano et al, Vol 109, Issue 9, 3849-3855
Methods Confocal Microscopy analysis. Cos-7 or HeLa cells were transfected with 2 µg of plasmids pEGFPC1 or p-EGFP-Nur77 DBD and 1 µg of plasmids pcDNA3-Myc-Bcl-2, pcDNA3-Myc-Bcl-B, pcDNA3-Myc-Bcl-XL, pcDNA3-Myc-Bfl-1, pcDNA3-Myc-Mcl-1 or pcDNA3-Bcl-W in the presence of 50 µM of z-VAD-fmk (Bachem). After 24 hrs, cells were fixed with PBS containing 3.7% formaldehyde and 2% sucrose for 15 min at room temperature, permeabilized with PBS containing 0.2% triton and 5% goat serum for 20 min at room temperature. Then, fixed cells were successively incubated with monoclonal anti-myc antibody (Roche) and goat anti-Hsp60 antibody (Santa-cruz) for 2 hrs at room temperature, washed with 0.1 % Triton in PBS, and then incubated with Texas red-conjugated rabbit-anti-mouse antibody and Cy-5 blue-conjugated rabbit-anti-goat antibody (Southern Biotechnology) for 1 hr at room temperature. Subcellular fractionation. Subcellular fractionation experiments were performed using ProteoExtract Subcellular Proteome extraction kit from Calbiochem according to the manufacturer’s instructions. The resulting fractions were analyzed by immunoblot using GFP and Myc antibodies and with Tubulin and Hsp60 antibodies, respectively, to verify cytosolic and mitochondrial fraction purity.
Files in this Data Supplement:
- Figure S1. Nur77 binds mouse Boo/Diva (PDF, 131 KB) -
293T cells in 6-well plates were cotransfected with 2 µg of plasmids encoding either GFP or GFP-Nur77ΔDBD in combination with 1 µg of plasmids encoding a Myc-tag version of either Bcl-2, Bcl-B, or Boo/Diva. After 24 hours, immunoprecipitation was performed using anti-Myc antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top) and anti-Myc (middle) antibodies. Cell lysates were also analyzed directly (50 µg) using anti-GFP antibody (bottom). Black and white arrowheads denote GFP-Nur77 and GFP proteins, respectively. Asterisks indicate nonspecific bands. The vertical line indicates the position at which extraneous lanes from the blot were digitally excised for clarity of presentation.
- Figure S2. Nur77 colocalizes in cells with Bcl-2, Bcl-B, and Bfl-1 (PDF, 133 KB) -
COS-7 cells were cotransfected with plasmids encoding GFP-Nur77DBD and Myc-tagged versions of various Bcl-2 family proteins. For Bcl-W we used a plasmid encoding a non-tagged Bcl-W protein. After 24 hours, cells were fixed, permeabilized, and successively incubated with anti-Myc or anti–Bcl-W antibodies followed by secondary antibodies conjugated to a red fluorochrome. Antibody localization was accomplished by confocal UV microscopy. Panels represent green (top), red (middle), and merge (bottom) channels.
- Figure S3. Nur77 is targeted to mitochondria when either Bcl-2 or Bcl-B is coexpressed (PDF, 1.96 MB) -
COS-7 cells were cotransfected with plasmids encoding GFP-Nur77DBD and control (“empty”) plasmid (top) or Myc-tagged versions of Bcl-2 (middle) or Bcl-B (bottom). After 24 hours, cells were fixed, permeabilized, and successively incubated with anti-Myc and Hsp60 (mitochondria staining) antibodies followed by secondary antibodies conjugated to a blue and red fluorochrome, respectively. Antibody localization was accomplished by confocal UV microscopy. Columns show green, blue, red, and merged channels (left to right, respectively).
- Figure S4. Bcl-B transmembrane domain is crucial for mitochondrial Nur77 localization (PDF, 47.3 KB) -
HeLa cells were cotransfected with plasmids encoding GFP-Nur77DBD and control plasmid (top) or plasmids encoding Myc-tagged Bcl-B (middle) or Bcl-BTM (bottom). After 24 hours, cells were fixed, permeabilized, and successively incubated with anti-Myc antibody followed by secondary antibody conjugated to a red fluorochrome. Antibody localization was accomplished by confocal UV microscopy. Columns represent green (left), red (middle), and merged (right) channels.
- Figure S5. Bcl-B transmembrane domain is crucial for mitochondrial Nur77 relocalization (PDF, 453 KB) -
HeLa cells were cotransfected with plasmids encoding either GFP or GFP-Nur77DBD and Myc-tagged versions of Bcl-B and Bcl-BTM. After 24 hours, cells were collected and subjected to subcellular fractionation. The resulting fractions were analyzed by immunobloting using anti-GFP, anti-Myc, antitubulin, and anti-Hsp60 antibodies.
- Figure S6. Immunohistochemical detection of Bcl-B in plasma cells (PDF, 295 KB) -
Tissue sections (gut lamina propria from a patient with Crohn disease) were double stained with the Bcl-B and CD138 antibodies using SG (black) for CD138 and DAB (brown) for Bcl-B. Nuclei are counterstained red. (A) The selected region was subjected to image analysis (red box). The black and brown colors were separated using a color deconvolution algorithm (Aperio) for CD138 (B) and Bcl-B (C). Results show Bcl-B as dark red pixels, with blue pixels corresponding to stained areas that are negative for Bcl-B. Note colocalization of CD138 and Bcl-B–positive cells. Original magnification ×|400.
- Figure S7. DC region of Nur77 is involved in Bcl-B binding (PDF, 75.8 KB) -
HEK293T cells were cotransfected with plasmids encoding either GFP or various GFP-Nur77 mutants in combination with plasmid encoding a Myc-tagged Bcl-B. After 24 hours, immunoprecipitation was performed using anti-Myc antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top) and anti-Myc (middle) antibodies. Cell lysates (50 µg) were also analyzed directly using anti-GFP antibody (bottom). The vertical line indicates the position at which extraneous lanes from the blot were digitally excised for clarity of presentation.
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