The Journal of Neuroscience, April 13, 2005, ():
c-Jun NH2-Terminal Kinase-Interacting Protein-3 Facilitates Phosphorylation and Controls Localization of Amyloid-
Precursor Protein
J. Neurosci. Muresan and Muresan
25: 3741
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Specific detection of pAPP by immunocytochemistry. Note that detection of pAPP is blocked by a phosphorylated (pACD; a, b), but not non-phosphorylated (ACD; c, d) polypeptide from the cytoplasmic domain of APP. Arrows show terminals of processes. (a, c) are corresponding phase-contrast micrographs. Only the left part of the field shown in (b) is shown in (a). Bar, 50 µm.
- supplemental material
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Supplemental Figure 2. Interaction of JIPs and Fe65 with APP. (a-d) Pull-down experiments done with a biotinylated polypeptide encompassing the APP cytoplasmic domain (ACD). Equal amounts of the polypeptide were incubated with lysates of COS-1 cells transfected with FLAG-tagged JIP-1, JIP-2, or JIP-3, or with myc-tagged Fe65, and collected on Streptavidin-Sepharose. Co-purified proteins were detected by immunoblotting with antibodies to the FLAG or myc tag. Note that JIP-3 fails to bind to the polypeptide. Samples of the inputs are shown at the left of each blot. Molecular size markers are in kDa.
- supplemental material
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Supplemental Figure 3. JIP-3-facilitated APP phosphorylation requires JNK activity. (a-f) Localization of pAPP in JIP-3-FLAG over-expressing CAD cells treated with either roscovitine (a-c) or SP600125 (d-f). CAD cells were labeled for pAPP (a, d) and FLAG (b, e). c, f are phase-contrast micrographs. Scale bar, 30 µm. (g) Quantitative measurement of the effect of roscovitine or SP600125 on JIP-3-facilitated APP phosphorylation. CAD cells were transfected with JIP-3-FLAG and treated with the inhibitors or DMSO (control). Percentages of transfected cells that showed increased pAPP levels are indicated. Note that the effect of kinase inhibitors on JIP-3-facilitated phosphorylation is similar to their effect on APP phosphorylation under normal conditions (see Fig. 2).
