The Journal of Neuroscience, August 2, 2006, ():
Induction of Autophagy in Axonal Dystrophy and Degeneration
J. Neurosci. Wang et al.
26: 8057
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. GFP-LC3 localization in soma and dendrites of Purkinje cells of GFP-LC3 transgenic (A) and GFP-LC3/Lurcher mice (B). (A) Distribution of GFP-LC3 fluorescence in Purkinje cells of GFP-LC3 transgenic (P20) was diffused. (B) An example of GFP-LC3 punctate structures in soma and dendrites of degenerating Purkinje cells in GFP-LC3/Lurcher mice (P20). Few GFP-LC3 puncta was seen in early postanatal day before P14. After P14, GFP-LC3/Lurcher mouse had a small fraction of the survived Purkinje cells expressing GFP-LC3 puncta in soma and dendrites. Scale bar: 20 μm.
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Supplemental Figure 2. Induction of autophagy as shown by partial inactivation of mTor kinase pathway in Lurcher cerebellum. Each lane was loaded with 40 μg proteins from cerebellar extracts of wild type (lane 1), Lurcher (lane 2), GFP-LC3 transgenic (lane 3) or GFP-LC3 transgenic crossed with Lurcher (lane 4) mouse (P13). Reduced phosphorylation of p70 S6 kinase (Thr412 phosphorylation for p85 and Thr 389 phosphorylation for p75) and S6 ribosomal protein (Ser 235/236 phosphorylation) in Lurcher samples (lanes 2 & 4) demonstrated the partial inactivation of mTor kinase pathway, which is consistent with the induction of autophagy. The mouse monoclonal phosphor-p70 kinase (Thr389) antibody and rabbit polyclonal p70 S6 kinase antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA) (1:200).
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Supplemental Figure 3. Identification of GFP-LC3-interacting proteins in GFP-LC3 transgenic mouse brains by Mass Spectrometry. (A) Three representative MS/MS spectra identified the high molecular weight protein band at lane 2 in Figure 4 as MAP1B. Parent masses were collected by a prototype QqTOF instrument. MS/MS spectra were collected by a LCQ ion trap instrument. MS/MS data were searched against NCBInr Mus musculus database using Xproteo. (B) A partial list of the GFP-LC3 interacting proteins identified in GFP-LC3 transgenic mouse brain by mass spectrometry.
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Supplemental Figure 4. Western blots showed that MAP1B(-P) is produced at equal amount in the brains of two Atg5-/- mice and their littermate Atg5+/+ (P0), demonstrating that MAP1B(-P) is not primarily degraded by autophagy. Equal amount of proteins was loaded on each lane.
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Supplemental Figure 5. Immunostaining of p62/SQSTM1 showed no fluorescence signal above background in axonal swelling of Lurcher Purkinje cells, suggesting that autophagosome accumulation seen in these axonal swellings is not due to impaired autophagy-lysosome degradation. The cerebellar slices of Lurcher mouse and its littermate (P13) were counterstained with anti-calbindin antibody to label the axonal dystrophic swelling of Lurcher Purkinje cells. Scale bar: 20 μm.
